CN1331187A - Polypeptide-human zinc finger protein 8.8 and polynudeotide for coding it - Google Patents

Polypeptide-human zinc finger protein 8.8 and polynudeotide for coding it Download PDF

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CN1331187A
CN1331187A CN 00116886 CN00116886A CN1331187A CN 1331187 A CN1331187 A CN 1331187A CN 00116886 CN00116886 CN 00116886 CN 00116886 A CN00116886 A CN 00116886A CN 1331187 A CN1331187 A CN 1331187A
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polypeptide
polynucleotide
zinc finger
sequence
finger protein
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毛裕民
谢毅
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上海博德基因开发有限公司
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Abstract

A novel polypeptide-human zinc finger protein 8.8, the polynucleotide for coding it, the process for preparing the said polypeptide by DNA recombination, the application of the said polypeptide in treating diseases such as embryo development teratogenesis, tumor, etc, the antagon of the said polypeptide and its medical action, and the application of the said polynucleotide are disclosed.

Description

一种新的多肽——人锌指蛋白8.8和编码这种多肽的多核苷酸 A new polypeptide - human zinc finger protein 8.8 and polynucleotides encoding such polypeptides

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——人锌指蛋白8.8,以及编码此多肽的多核苷酸序列。 The present invention belongs to the field of biotechnology, in particular, the present invention describes a new polypeptide - human zinc finger protein 8.8, and the polynucleotide sequence encoding the polypeptide. 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 also relates to methods and apply polynucleotides and polypeptides.

因的转录调控对于基因的正常表达及发挥生物学功能是十分重要的,通常由转录调控因子来完成这一过程。 Transcriptional regulation because of the normal biological function of gene expression and play is very important, usually done by a transcriptional regulator of this process. 转录调控因子在生物体内参与决定基因在何种组织及何种发育阶段开始转录,编码这类蛋白的基因如发生突变,不但该基因自身不能正常表达,而且受其调节的许多基因也不能正常的进行转录与表达。 Transcriptional regulatory factors involved in determining what tissue in vivo and what developmental stage transcription start, a gene encoding such protein, such as mutations, not only normal expression of the gene itself, but also modulated by a number of genes not in the normal transcription and expression. 转录因子对基因表达的调控主要通过转录因子与特定的DNA序列结合、转录因子间的相互作用及转录因子与常规转录机构的相互作用在完成。 Transcription factor to regulate gene expression through transcription factors primarily binding to specific DNA sequences, transcription factors and the interaction between the conventional mechanism of transcription transcription factor interactions completed. 根据结构基序的不同,已知的DNA结合蛋白可主要分为两类:含有螺旋-转角-螺旋基序的蛋白及锌指蛋白[Kamal Chowdhury,Heidi Rohdewohld et a1.,Nucleic Acids Research,1988,16:9995-10011]。 The different structural motifs, known DNA binding proteins can be divided into two categories: comprising helix - turn - helix motif and the zinc finger protein proteins [Kamal Chowdhury, Heidi Rohdewohld et a1, Nucleic Acids Research, 1988, 16: 9995-10011].

锌指蛋白为编码锌离子介导的核苷酸结合蛋白多基因家族中的成员,锌指蛋白的锌指结构主要有以下几种:C2H2构型、C2C2构型、C2HC构型、C2HC4C构型、C3H构型、C3HC4构型(Dai KS et al.,1998)。 Zinc finger protein encoding nucleotide zinc ion-mediated binding member multigene family proteins, zinc finger proteins Zinc finger are the following: C2H2 configuration, C2C2 configuration, C2HC configuration, C2HC4C configuration , C3H configuration, C3HC4 configuration (Dai KS et al., 1998). 人们已从酵母、果蝇、鼠及人等多种生物体中分离得到了各种构型的锌指蛋白,其中含C2H2构型的锌指蛋白基因构成人类基因组最大的一族基因(Berker et al.,1995)。 People from yeast, Drosophila, mice and other organisms who isolated a various configurations zinc finger protein, which contains configuration C2H2 zinc finger protein genes constitute the largest family of human genome genes (Berker et al ., 1995). C2H2型锌指蛋白与基因的转录活化及抑制有关,这些蛋白的表达异常将引发各种发育紊乱性疾病、各种肿瘤的发生、各种遗传性疾病及免疫系统疾病[KamalChowdhury,Heidi Rohdewohld et al.,Nucleic Acids Research,1988,16:9995-10011]。 C2H2 zinc finger transcription activator protein and the inhibition of gene expression of these proteins will cause various abnormal developmental disorders, the occurrence of a variety of tumors, various genetic diseases and immune system disorders [KamalChowdhury, Heidi Rohdewohld et al ., Nucleic Acids Research, 1988,16: 9995-10011].

所有的C2H2型锌指蛋白均含有28-30个氨基酸长的保守的指重复序列(F/Y)XCXXCXXXFXXXXXLXXHXXXHTGEKP,其中一些特定的氨基酸残基位点为高度保守的。 All C2H2 zinc finger proteins contain conserved 28-30 amino acid long finger repeats (F / Y) XCXXCXXXFXXXXXLXXHXXXHTGEKP, wherein some specific amino acid residue positions that are highly conserved. 这一序列在很多不同的锌指蛋白中均含有多个拷贝,其拷贝数不同(锌指个数不同)则功能也不同。 The sequence in many different zinc finger proteins contain multiple copies of different copy number (the number of different zinc finger) is different functions. 锌指蛋白与不同长度的DNA的结合依赖于指结构的数量,多指结构可能与复合物的结合稳定性有关,而复合物是RNA聚合酶转录的作用位点。 Zinc finger DNA binding protein to the number of different lengths dependent on the finger, the finger may be associated with multiple binding stability of the complex, the complex is an RNA polymerase transcribes the site of action. 研究发现,许多锌指蛋白的锌指结构域相互连接区域也是高度保守的,这一区域通常含有下列序列:His-Thr-Gly-Gly-Lys-Pro-(Tyr,Phe)-X-Cys,其中组氨酸与半胱氨酸为金属离子的结合位点,而X为可变氨基酸残基。 The study found that many of the zinc finger proteins connected to each other zinc finger domains is highly conserved regions, this region usually contains the following sequence: His-Thr-Gly-Gly-Lys-Pro- (Tyr, Phe) -X-Cys, wherein the cysteine ​​and histidine binding site of the metal ion, and X is a variable amino acid residues. 这一区域对于锌指结构的形成是必需的,指结构的数量将直接影响锌指蛋白与不同长度的DNA结合,且多指结构与转录调节复合物的结合稳定性有关[JeremyM.Berg,Annu.Rev.Biophys.Chem,1990,19:405-421]。 The region for forming a zinc finger structure are required, the number refers to the structure will directly affect the zinc finger protein DNA binding of different lengths, and the multi-finger structure and stability of the complex in conjunction with transcriptional regulatory related [JeremyM.Berg, Annu .Rev.Biophys.Chem, 1990,19: 405-421]. 按锌指蛋白蛋白序列的特征,还可将C2H2型锌指蛋白分为各种不同的蛋白亚家族,这些蛋白亚家族的成员的氨基酸序列中亦含有如上所述的保守序列片段,并在生物体内具有相似的功能特征。 Wherein the zinc finger protein by protein sequence, will also C2H2 zinc finger protein into different subfamily, the amino acid sequences of the members of the subfamily of these proteins contain a conserved sequence fragment also described above, biological and having similar functional characteristics in vivo.

1997年,Shi等人又从人中克隆得到了锌指蛋白家族的一个新成员ZNF191。 In 1997, Shi et al., And from among cloned a new zinc finger protein family ZNF191. 该蛋白的氨基酸序列中亦含有如上所述的保守的一致性序列片段,且其在生物体造血细胞、脑、神经系统、表皮组织、各种与分泌吸收相关的组织及与肿瘤和无限增殖细胞系相关的组织中均有表达。 The amino acid sequence of the protein also contains a conserved consensus sequence fragments described above, and associated with tumor tissue and immortalized cell and its absorption in the organism of hematopoietic cells, brain, nervous system, epidermis, and secrete various Department-related organizations were expressed. 因而,该家族的成员对生物体内各种组织的分化及发育都起着十分重要的作用。 Thus, members of this family of growth and differentiation of various tissues in vivo plays a very important role. 它们在生物体内可有效地控制各种基因的转录表达水平,其表达异常可能会导致细胞的异常分化及增殖,从而引发各种疾病,如癌症及各种免疫系统疾病。 They are effective in controlling a variety of in vivo expression level of a gene transcript, the abnormal expression of which may lead to abnormal differentiation and proliferation of cells, causing a variety of diseases, such as cancer and various immune system disorders. 该蛋白在生物体内通常与一些免疫系统疾病、神经系统疾病、各种炎症反应等的发生密切相关。 The protein is typically closely associated with a number of immune system diseases, neurological diseases, various inflammatory reactions in vivo. 其还可用于诊断及治疗上述各种相关的疾病。 It may also be used for diagnosis and treatment of various diseases related above.

通过基因芯片的分析发现,在膀胱粘膜、PMA+的Ecv304细胞株、LPS+的Ecv304细胞株胸腺、正常成纤维细胞1024NC、Fibroblast,生长因子刺激,1024NT、疤痕成fc生长因子刺激,1013HT、疤痕成fc未用生长因子刺激,1013HC、膀胱癌建株细胞EJ、膀胱癌旁、膀胱癌、肝癌、肝癌细胞株、胎皮、脾脏、前列腺癌、空肠腺癌、贲门癌中,本发明的多肽的表达谱与人锌指蛋白的表达谱非常近似,因此二者功能也可能类似。 By analyzing microarray found in the bladder mucosa, PMA + of Ecv304 cell lines, LPS + of Ecv304 cell lines thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scars into fc growth factor stimulation, 1013HT, scars into fc not stimulated with growth factors 1013HC, cultured cells of bladder cancer EJ, adjacent bladder cancer, bladder cancer, hepatoma, hepatocellular carcinoma cell lines, fetal skin, spleen, prostate cancer, adenocarcinoma of the jejunum, cardia, the expression of the polypeptide of the present invention. spectrum of human zinc finger protein expression profile very similar, so the two functions may be similar. 本发明被命名为人锌指蛋白8.8。 The present invention has been named human zinc finger protein 8.8.

由于如上所述人锌指蛋白8.8蛋白在调节细胞分裂和胚胎发育等机体重要功能中起重要作用,而且相信这些调节过程中涉及大量的蛋白,因而本领域中一直需要鉴定更多参与这些过程的人锌指蛋白8.8蛋白,特别是鉴定这种蛋白的氨基酸序列。 As described above since the human zinc finger protein 8.8 Protein body plays an important role in regulating cell division and vital functions of embryonic development and the like, and believe that a large number of proteins involved in these regulatory processes, and the present art has been the need to identify more involved in these processes 8.8 human zinc finger protein proteins, especially the amino acid sequence identified this protein. 新人锌指蛋白8.8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。 8.8 zinc finger protein isolate new gene encoding this protein is also determined studies provided the basis role in health and disease. 这种蛋白可能构成开发疾1病诊断和/或治疗药的基础,因此分离其编码DNA是非常重要的。 This protein may constitute a diagnosis and / or therapeutic agent based on the development of the disease, its coding DNA thus isolated is very important.

本发明的一个目的是提供分离的新的多肽——人锌指蛋白8.8以及其片段、类似物和衍生物。 An object of the present invention to provide a new an isolated polypeptide - human zinc finger protein 8.8 as well as fragments, analogs and derivatives.

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

本发明的另一个目的是提供含有编码人锌指蛋白8.8的多核苷酸的重组载体。 Another object of the present invention is to provide a zinc finger comprising a polynucleotide encoding a recombinant carrier protein 8.8.

本发明的另一个目的是提供含有编码人锌指蛋白8.8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 zinc finger comprising a polynucleotide encoding a genetically engineered host cell protein 8.8.

本发明的另一个目的是提供生产人锌指蛋白8.8的方法。 Another object of the present invention is to provide a human zinc finger protein production methods 8.8.

本发明的另一个目的是提供针对本发明的多肽——人锌指蛋白8.8的抗体。 Another object of the present invention to provide for a polypeptide of the invention - human zinc finger protein antibody 8.8.

本发明的另一个目的是提供了针对本发明多肽——人锌指蛋白8.8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 polypeptide of the invention for - human zinc finger protein analog compounds 8.8, antagonists, agonists, inhibitors.

本发明的另一个目的是提供诊断治疗与人锌指蛋白8.8异常相关的疾病的方法。 Another object of the present invention is to provide a diagnostic method for the treatment of abnormal human zinc finger protein-related diseases 8.8.

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。 The present invention relates to an isolated polypeptide, the polypeptide is of human origin, which comprises: a polypeptide having the amino acid sequence of SEQ ID No.2, or a conservative variant thereof, a biologically active fragment or derivative thereof. 较佳地,该多肽是具有SEQ ID NO:2氨基酸序列的多肽。 Preferably, the polypeptide having SEQ ID NO: 2 amino acid polypeptide sequence.

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:(a)编码具有SEQ ID No.2氨基酸序列的多肽的多核苷酸;(b)与多核苷酸(a)互补的多核苷酸;(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。 The present invention further relates to an isolated polynucleotide which comprises a nucleotide sequence selected from the group consisting of or variants thereof: (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No.2; ( b) a polynucleotide of (a) a polynucleotide complementary; (c) to (a) or (b) a polynucleotide sequence having at least 70% identical to a polynucleotide.

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80-464位的序列;和(b)具有SEQ ID NO:1中1-716位的序列。 More preferably, the polynucleotide is a sequence selected from the group: (a) having SEQ ID NO: 1 in the sequence of positions 80-464; and (b) having the SEQ ID NO: 1 in the 1-716 bit sequence.

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

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

本发明还涉及一种筛选的模拟、激活、拮抗或抑制人锌指蛋白8.8蛋白活性的化合物的方法,其包括利用本发明的多肽。 The present invention further relates to a method for screening analog, activation, antagonistic compound, or human zinc finger protein 8.8 methods of inhibiting the activity of a protein, which comprises using the polypeptide of the present invention. 本发明还涉及用该方法获得的化合物。 The present invention also relates to compounds obtained by this method.

本发明还涉及一种体外检测与人锌指蛋白8.8蛋白异常表达相关的疾病或疾病易感性的方法,包括检测生物样品中所述多肽或其编码多核苷酸序列中的突变,或者检测生物样品中本发明多肽的量或生物活性。 The present invention further relates to an in vitro method of detecting human zinc finger protein 8.8 abnormal expression associated with a disease or disease susceptibility, a biological sample comprising detecting the polypeptide or a polynucleotide encoding a mutant sequence, or a biological sample amount or the biological activity of the polypeptide of the present 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 present invention also relates to a pharmaceutical composition containing the polypeptide of the invention or a mimetic, activator, inhibitor, or antagonist 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人锌指蛋白8.8表达异常所引起疾病的药物的用途。 The present invention further relates to a polypeptide of the invention and / or polynucleotides for the treatment of cancer, developmental disease or autoimmune disease or other drug due to the expression of human zinc finger protein 8.8 diseases caused by abnormal in.

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。 Other aspects of the present invention, since the techniques disclosed herein, to those skilled in the art will be apparent.

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。 The following terms used in the specification and claims unless otherwise specified have the following meanings: "nucleic acid sequence" refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, may also be of genomic or synthetic DNA or RNA, which may be single or double stranded and represent the sense strand or the antisense strand. 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。 Similarly, the term "amino acid sequence" means an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof. 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。 When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, this "polypeptide" or "protein" are not meant to limit the amino acid sequence associated with the protein molecule intact native amino acid .

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。 Protein or polynucleotide "variant" refers to an amino acid having one or more nucleotides or amino acid sequence or altered polynucleotide sequence encoding it. 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。 The changes may include deletion of an amino acid or nucleotide sequence or nucleotide sequences, insertion or substitution. 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。 Variant may have "conservative" changes, wherein a substituted amino acid has similar primary amino acid structure or chemical properties, such as replacement of leucine with isoleucine. 变体也可具有非保守性改变,如用色氨酸替换甘氨酸。 Variant may also have non-conservative changes, such as replacing glycine with a 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" means deletion of one amino acid sequence or nucleotide sequence, or more amino acids or nucleotides.

“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。 "Insert" or "Add" refers to an amino acid sequence or nucleotide sequence changes result compared to naturally occurring molecules, one or more amino acids or to increase nucleotides. “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。 "Replacement" refers to replacing one or more amino acids or nucleotides by 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。 "Biological activity" refers to a protein structure, regulatory or biochemical function of the natural molecule. 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。 Similarly, the term "immunologically active" refers to the ability of the natural, recombinant or synthetic proteins and fragments induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

“激动剂”是指当与人锌指蛋白8.8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。 "Agonist" refers to a time when the human zinc finger protein binding 8.8 A can cause changes to adjust the protein molecules of the protein activity. 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人锌指蛋白8.8的分子。 Agonist may include proteins, nucleic acids, carbohydrates, or any other human zinc finger protein binding 8.8 molecules.

“拮抗剂”或“抑制物”是指当与人锌指蛋白8.8结合时,一种可封闭或调节人锌指蛋白8.8的生物学活性或免疫学活性的分子。 "Antagonist" or "inhibitor" refers to human zinc finger protein 8.8 when binding a closable regulating human zinc finger molecule or biological activity or immunological activity of protein 8.8. 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人锌指蛋白8.8的分子。 Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule binding to human zinc finger protein 8.8.

“调节”是指人锌指蛋白8.8的功能发生改变,包括蛋白质活性的升高或降低、结合特性的改变及人锌指蛋白8.8的任何其它生物学性质、功能或免疫性质的改变。 "Modulation" refers to a human zinc finger protein 8.8 functional changes, including increase or decrease in protein activity, binding characteristics change and human zinc finger proteins alter any other biological properties of 8.8, function, or immunological properties.

"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。 "Substantially pure" means substantially free of naturally associated with other proteins, lipids, carbohydrates or other substances. 本领域的技术人员能用标准的蛋白质纯化技术纯化人锌指蛋白8.8。 Those skilled in the art can be purified by standard protein purification techniques human zinc finger protein 8.8. 基本上纯的人锌指蛋白8.8在非还原性聚丙烯酰胺凝胶上能产生单一的主带。 Substantially pure human zinc finger protein 8.8 can produce a single major band on a non-reducing polyacrylamide gel. 人锌指蛋白8.8多肽的纯度可用氨基酸序列分析。 8.8 human zinc finger protein purity available polypeptide amino acid sequence analysis.

“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。 "Complementary" or "complementarity" refers to a polynucleotide naturally at a salt concentration and temperature conditions by base pairing to allow binding. 例如,序列“CTGA”可与互补的序列“GACT”结合。 For example, the sequence "CTGA" can be combined with a sequence complementary "GACT". 两个单链分子之间的互补可以是部分的或全部的。 Between two complementary single-stranded molecules may be partial or total. 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。 The degree of complementarity between nucleic acid strands has significant effects 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。 "Homology" refers to the degree of complementarity, and may be partial homology or complete homology. “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。 "Partially homologous" refers to a partial sequence complementary to its target nucleic acid sequence to hybridize to at least partially inhibits a completely complementary. 这种杂交的抑制可通过在严格性程度降低的条件下进行杂交(Southern印迹或Northern印迹等)来检测。 Such inhibition may be by hybridization under reduced stringency hybridization conditions degree (Southern blot or Northern blot, etc.) is detected. 基本上同源的序列或杂交探针可竞争和抑制完全同源的序列与靶序列在的严格性程度降低的条件下的结合。 Substantially homologous sequence or hybridization probe may compete for and inhibit the binding of a completely homologous sequence to reduce the target sequence in the degree of stringency conditions. 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。 It is not intended to reduce the degree of stringency conditions that allow non-specific binding, since the degree of reduced stringency conditions require binding of two sequences to each other 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。 "Percent identity" refer to the same or similar in the two or more amino acid sequence or nucleic acid sequence comparison in percentage. 可用电子方法测定相同性百分率,如通过MEGALIGN程序(Lasergenesoftware package,DNASTAR,Inc.,Madison Wis.)。 Available electronically measuring the percentage identity, as determined by MEGALIGN program (Lasergenesoftware package, DNASTAR, Inc., Madison Wis.). MEGALIGN程序可根据不同的方法如Cluster法比较两种或多种序列(Higgins,DG和PMSharp(1988)Gene 73:237-244)。 The MEGALIGN program can compare two or more sequences (Higgins, DG and PMSharp (1988) Gene 73: 237-244) Cluster Method according to different methods. Cluster法通过检查所有配对之间的距离将各组序列排列成簇。 Cluster method by examining the distances between all pairs of each set of sequence arranged in clusters. 然后将各簇以成对或成组分配。 Each cluster is then assigned in pairs or in groups. 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:序列A与序列B之间匹配的残基个数 Two percent amino acid sequence identity between the sequences of sequences A and B calculated by the formula: The number of residue matches between sequence A and sequence B 100序列A的残基数—序列A中间隔残基数—序列B中间隔残基数也可以通过Cluster法或用本领域周知的方法如Jotun Hein测定核酸序列之间的相同性百分率(Hein J.,(1990)Methods in emzumology 183:625-645)。 A sequence of 100 residues - residues in sequence A interval - interval B sequence residues may be present by methods known in the art such as the Jotun Hein measuring the percentage identity between nucleic acid sequences (Hein J Cluster method or by ., (1990) Methods in emzumology 183: 625-645).

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

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

“衍生物”是指HFP或编码其的核酸的化学修饰物。 "Derivative" refers to a chemical modification or a nucleic acid encoding the HFP thereof. 这种化学修饰物可以是用烷基、酰基或氨基替换氢原子。 Such chemical modification may be a replacement of a hydrogen atom with an alkyl, acyl, or amino. 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。 Nucleic acid derivatives may retain biological properties major polypeptide molecule encoding naturally.

“抗体”是指完整的抗体分子及其片段,如Fa、F(ab')2及Fv,其能特异性结合人锌指蛋白8.8的抗原决定簇。 "Antibody" refers to an intact antibody molecules and fragments thereof, such as Fa, F (ab ') 2 and Fv, which are capable of specifically binding to human zinc finger protein 8.8 antigenic determinants.

“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。 "Humanized antibody" refers to an amino acid sequence of the non-antigen binding regions are replaced with human antibody become more similar, but still retaining the original binding activity of the antibody.

“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。 "Isolated" refers to material from its original environment (e.g., if it refers to naturally occurring in its natural environment) being removed. 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。 For example, a polynucleotide or polypeptide naturally occurring in a living animal is present is not isolated, but the same polynucleotide or polypeptide with some or all coexist in the natural system, is isolated separate substances. 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。 Such polynucleotides could be part of a vector, such polynucleotides may be part of a polypeptide or composition. 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。 Since the vector or composition is not a component of its natural environment that they remain separate.

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)。 As used in this invention, "isolated" refers to material isolated from its original environment out (if it is a natural substance, i.e., the original environment is the natural environment). 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。 The polynucleotide and polypeptide in the native state in living cells are not isolated and purified, but the same polynucleotide or polypeptide separated from such other substances that exist in the natural state, the separation and purification compared with the .

如本文所用,“分离的人锌指蛋白8.8”是指人锌指蛋白8.8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。 As used herein, "isolated human zinc finger protein 8.8" refers to a human zinc finger protein 8.8 substantially free of naturally associated with other proteins, lipids, carbohydrates or other substances. 本领域的技术人员能用标准的蛋白质纯化技术纯化人锌指蛋白8.8。 Those skilled in the art can be purified by standard protein purification techniques human zinc finger protein 8.8. 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。 Substantially pure polypeptide capable of producing a single major band on a non-reducing polyacrylamide gel. 人锌指蛋白8.8多肽的纯度能用氨基酸序列分析。 Analysis of human zinc finger protein purity can be 8.8 amino acid sequence of the polypeptide.

本发明提供了一种新的多肽——人锌指蛋白8.8,其基本上是由SEQ ID NO:2所示的氨基酸序列组成的。 The present invention provides a novel polypeptide - human zinc finger protein 8.8, consisting essentially of SEQ ID NO: 2 amino acid sequence thereof. 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。 Polypeptides of the invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. 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。 Polypeptides of the invention may be a naturally purified product, or a product of chemical synthesis, or using recombinant techniques (e.g., bacterial, yeast, higher plant, insect and mammalian cells) produced from a prokaryotic or eukaryotic host. 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。 The recombinant production, host employed, polypeptides of the invention may be glycosylated or may be non-glycosylated. 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。 Polypeptides of the invention may or may not include a starting methionine residue.

本发明还包括人锌指蛋白8.8的片段、衍生物和类似物。 The present invention further comprises a fragment of human zinc finger protein 8.8, derivatives and analogs thereof. 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锌指蛋白8.8相同的生物学功能或活性的多肽。 As used herein, the term "fragment", "derivative" and "analog" refers to the present invention remains substantially human zinc finger polypeptide with the same biological function or activity of protein 8.8. 本发明多肽的片段、衍生物或类似物可以是:(I)这样一种,其中一个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代,并且取代的氨基酸可以是也可以不是由遗传密码子编码的;或者(II)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(III)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、00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。 Polypeptide fragments of the present invention, derivatives or analogs may be: (I) one in which one or more amino acid residues are substituted with conservative or non-conserved amino acid residue (preferably a conserved amino acid residue), and a substituted amino acids may or may not encoded by the genetic code; or (II) a, wherein a group of one or more amino acid residue is substituted with another substituent containing groups; or (III) so one of which the mature polypeptide with another compound (such as extended half-life of polypeptide compound, such as polyethylene glycol); or (IV) a, wherein the additional polypeptide sequence into the amino acid sequence of the mature polypeptide fused formed ( such as a leader or secretory sequence or a sequence for purification of the polypeptide or a proprotein sequence) by the forth herein such fragments, derivatives and analogs 00 are considered within the knowledge of the skilled artisan.

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。 The present invention provides an isolated nucleic acid (polynucleotide), having a substantially encoded by SEQ ID NO: 2 amino acid polypeptide polynucleotide sequence. 本发明的多核苷酸序列包括SEQ ID NO:1的核苷酸序列。 The polynucleotide sequences of the invention include SEQ ID NO: 1 is the nucleotide sequence. 本发明的多核苷酸是从人胎脑组织的cDNA文库中发现的。 Polynucleotides of the present invention is the discovery of a cDNA library from human fetal brain tissue. 它包含的多核苷酸序列全长为716个碱基,其开放读框80-464编码了80个氨基酸。 It contains the full length polynucleotide sequence of 716 bases, an open reading frame which encodes a 80 amino acid 80-464. 根据基因芯片表达谱比较发现,此多肽与人锌指蛋白有相似的表达谱,可推断出该人锌指蛋白8.8具有人锌指蛋白相似的功能。 The gene chip expression profile comparison, this polypeptide with human zinc finger proteins have similar expression profiles, can be inferred that the person who has 8.8 zinc finger protein similar to zinc finger protein function.

本发明的多核苷酸可以是DNA形式或是RNA形式。 Polynucleotide of the invention may be in the form of DNA or RNA form. DNA形式包括cDNA、基因组DNA或人工合成的DNA。 The form of DNA including cDNA, genomic DNA or synthetic DNA. DNA可以是单链的或是双链的。 DNA may be single-stranded or double-stranded. DNA可以是编码链或非编码链。 DNA may be the coding strand or non-coding strand. 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。 Encoding the mature polypeptide coding region sequence may be SEQ ID NO: same as the coding sequence shown in Figure 1 or a degenerate variant thereof. 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。 As used in this invention, "degenerate variants thereof" in the present invention refers to encoding a SEQ ID NO: 2 is a protein or polypeptide, but SEQ ID NO: 1 coding region of the nucleic acid sequence shown in sequence differences.

编码SEQ ID NO:2的成熟多肽的多核苷酸包括:只有成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。 Encoding SEQ ID NO: 2 of the mature polypeptide comprises: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequence; the coding sequence of the mature polypeptide (and optionally additional coding sequence) and non- 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" is meant to include a polynucleotide encoding said polypeptide and polynucleotides includes additional coding and / or non-coding sequence.

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。 The present invention further relates to the above described variants of the polynucleotide, polypeptide or polypeptide fragment has the same amino acid sequence, which encodes the analogues and derivatives of the present invention. 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。 This variant of the polynucleotide may be a variant allelic variants of naturally occurring or non-naturally occurring. 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。 These nucleotide variants include substitution variants, deletion variants, and insertion variants. 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。 As known in the art, an allelic variant is an alternate form of a polynucleotide, it may be one or more nucleotide substitutions, deletions or insertions, but does not materially change the function of the encoded polypeptide .

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)。 (At least 50% between the two sequences, preferably 70% identical to) the present invention also relates to a polynucleotide sequence hybridizing to the above-described. 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。 The present invention particularly relates to polynucleotides under stringent conditions to the polynucleotide of the present invention can hybridize. 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0.2×SSC,0.1%SDS,60℃;或(2)杂交时加用变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃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。 In the present invention, "stringent conditions" refers to: (1) hybridization and wash under lower ionic strength and higher temperature, such as 0.2 × SSC, 0.1% SDS, 60 ℃; or (2) hybridization plus with a denaturant, such as 50% (v / v) formamide, 0.1% calf serum /0.1%Ficoll,42℃ the like; or (3) only identity between two sequences of at least 95%, more good hybridization occurs when more than 97%. 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。 Further, the polynucleotide encoding the polypeptide hybridize with SEQ ID NO: 2 shown in the mature polypeptide having the same biological function and activity.

本发明还涉及与以上所描述的序列杂交的核酸片段。 The present invention further relates to nucleic acid fragments hybridize with the sequence described above. 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。 As used in this invention, "nucleic acid fragment" having a length of at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, preferably at least 100 nuclei nucleotide or more. 核酸片段也可用于核酸的扩增技术(如PCR)以确定和/或分离编码人锌指蛋白8.8的多核苷酸。 Nucleic acid fragments may also be used in nucleic acid amplification techniques (e.g. PCR) to determine and / or isolate polynucleotides encoding zinc finger protein 8.8.

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。 The polypeptides and polynucleotides of the present invention are preferably provided in isolated form, more preferably purified to homogeneity.

本发明的编码人锌指蛋白8.8的特异的多核苷酸序列能用多种方法获得。 The present invention encoding zinc finger proteins specific polynucleotide sequences can be 8.8 to obtain a variety of methods. 例如,用本领域熟知的杂交技术分离多核苷酸。 For example, well known in the art isolated polynucleotide hybridization techniques. 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。 These techniques include, but are not limited to: 1) a polynucleotide probe sequences of genomic or cDNA libraries with hybridization to homologous detected, and 2) the expression detection antibody libraries screened clones having the common structural features of the polynucleotide acid fragment.

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。 DNA fragments of sequences of the invention can be also obtained the following methods: 1) Double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of double stranded DNA to obtain DNA sequence the polypeptide.

上述提到的方法中,分离基因组DNA最不常用。 The method mentioned above, the genomic DNA was isolated most unusual. DNA序列的直接化学合成是经常选用的方法。 Direct chemical synthesis of DNA sequences is frequently the method of choice. 更经常选用的方法是cDNA序列的分离。 The method chosen more frequently isolated cDNA sequence. 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。 CDNA isolated by standard methods from the high expression of interest is the gene donor cells and reverse transcribed mRNA isolated form plasmid or bacteriophage cDNA library. 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)。 The method of extracting mRNA has a variety of mature technology, the kit also available (Qiagene) from commercial sources. 而构建cDNA文库也是通常的方法(Sambrook,et al.,MolecularCloning,A Laboratory Manual,Cold Spring Harbor Laboratory.New York,1989)。 CDNA library is constructed in a conventional method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989). 还可得到商业供应的cDNA文库,如Clontech公司的不同cDNA文库。 It can also be obtained commercially available cDNA libraries, such as the different cDNA libraries from Clontech. 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。 When used in conjunction with a polymerase reaction technology, even rare 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。 Using conventional screening method of gene of the present invention from these cDNA libraries. 这些方法包括(但不限于):(1)DNA-DNA或DNA-RNA杂交;(2)标志基因功能的出现或丧失;(3)测定人锌指蛋白8.8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。 These methods include (without limitation) :( 1) DNA-DNA or DNA-RNA hybridization; (2) a marker gene function or loss occurs; (3) measuring human zinc finger protein transcript levels of 8.8; (4) by immunological techniques or biological activity assay to detect the protein products of gene expression. 上述方法可单用,也可多种方法联合应用。 The method described above can be used alone, it can also be combined application of numerous approaches.

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。 In the (1) method, the hybridized probe is used with a polynucleotide homologous to any part of the present invention, a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, preferably at least 100 nucleotides. 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。 Further, generally in the length of the probe of 2000 nucleotides, preferably within 1000 nucleotides. 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。 As used herein, probes based on the gene sequence are usually present invention, information on the chemical synthesis of DNA sequences. 本发明的基因本身或者片段当然可以用作探针。 Or a fragment of the gene itself of the present invention may of course be used as probes. 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。 Labeled DNA probe may be radiolabeled, fluorescein or an enzyme (such as alkaline phosphatase) and the like.

在第(4)种方法中,检测人锌指蛋白8.8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, the detection of human zinc finger protein gene 8.8 protein product available immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and the like.

应用PCR技术扩增DNA/RNA的方法(Saiki,et al.Science1985;230:1350-1354)被优选用于获得本发明的基因。 The method (Saiki, et al.Science1985; 230: 1350-1354) by PCR amplification of DNA / RNA is preferred for obtaining the gene of the present invention. 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。 Particularly difficult to obtain full-length cDNA from the library, RACE method can be preferably used (-RACE rapid amplification of cDNA end), for PCR primers suitable information according to the present invention the polynucleotide sequences disclosed herein selected and synthesized by conventional methods. 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。 Standard methods such as gel electrophoresis and purified by the amplified DNA / RNA fragment.

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。 The polynucleotide sequence of the gene of the present invention obtained as described above, the various DNA fragments or the like by conventional methods such as the dideoxy chain termination method (Sanger et al.PNAS, 1977,74: 5463-5467) was measured. 这类多核苷酸序列测定也可用商业测序试剂盒等。 Such polynucleotide sequences can also be measured commercial sequencing kit and the like. 为了获得全长的cDNA序列,测序需反复进行。 To obtain the full-length cDNA sequences, which are repeated sequencing. 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。 Sometimes necessary to measure a plurality of cloned cDNA sequences can be assembled into 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锌指蛋白8.8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 with the vector of the invention or host cells directly with the human zinc finger protein 8.8 coding sequence produced by genetic engineering, and methods of producing the polypeptides of the invention by recombinant techniques.

本发明中,编码人锌指蛋白8.8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。 In the present invention, encoding a zinc finger protein 8.8 polynucleotide sequence may be inserted into the vector, to form recombinant vector comprising the polynucleotide of the present invention. 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。 The term "carrier" refers to known in the art bacterial plasmids, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors. 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。 Suitable carriers in this invention include, but are not limited to: expression in bacteria of the T7 promoter expression vector (Rosenberg, et al.Gene, 1987,56: 125) based; pMSXND expression in a mammalian cell expression vector ( lee and Nathans, J Bio Chem.263: 3521,1988) and baculovirus-derived vectors for expression in insect cells. 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。 In short, as long as the recombinant expression vector and stable replication in the host, any plasmids and vectors can be used to construct. 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。 An important feature of the expression vector typically contains an origin of replication, promoter, marker gene and translation regulation element.

本领域的技术人员熟知的方法能用于构建含编码人锌指蛋白8.8的DNA序列和合适的转录/翻译调控元件的表达载体。 Those skilled in the art well known methods can be used to construct encoding a zinc finger-containing protein expression vector a DNA sequence of 8.8 and appropriate transcriptional / translational control elements. 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 LaboratoryManual,cold Spring Harbor Laboratory.New York,1989)。 These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant technology (Sambroook, et al.Molecular Cloning, a LaboratoryManual, 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。 The DNA sequence may be operably linked to an appropriate promoter in the expression vector, to direct mRNA synthesis. 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。 Representative examples of such promoters include: E. coli lac or trp promoter; [lambda] phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, expression retrovirus LTRs and other genes known to be controlled in prokaryotic cells or eukaryotic cells or their viruses promoter. 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。 The expression vector further comprises a translation initiation by a ribosome binding site and a transcription terminator and the like. 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。 Inserting an enhancer sequence into the vector it will be transcribed in higher eukaryotic cells is enhanced. 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。 Enhancers are cis-acting DNA element expressed, usually about 10 to 300 base pairs, act on a promoter to increase transcription of the gene. 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。 Examples cited include the late side of the replication origin 100-270 bp SV40 enhancer, adenovirus enhancer and the enhancer on the late side of the replication origin polyoma like.

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 vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as eukaryotic cell culture with dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP), for E. coli, or tetracycline or ampicillin resistance and the like.

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 an appropriate vector / selectable marker gene and transcriptional regulatory elements (e.g., promoters, enhancers, etc.).

本发明中,编码人锌指蛋白8.8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。 In the present invention, a polynucleotide encoding a zinc finger protein 8.8 or recombinant vector containing the polynucleotide may be transformed or transfected into host cells to genetically engineered host cells constituting a recombinant containing the polynucleotide or vector. 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。 The term "host cell" refers to a prokaryotic cell, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. 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CHO、COS或Bowes黑素瘤细胞等。 Representative examples are: E.coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, 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。 With a DNA sequence according to the present invention or a recombinant vector comprising the DNA sequence of transforming a host cell by conventional techniques well known to those skilled performed. 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。 When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be in exponential growth phase were harvested after treatment with CaCl2 method, the steps used are well known in the art. 可供选择的是用MgCl2。 Alternative is to use MgCl2. 如果需要,转化也可用电穿孔的方法进行。 If desired, the conversion process may be performed using electroporation. 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。 When the host is a eukaryote, optional DNA transfection methods as follows: calcium phosphate coprecipitation method, or conventional mechanical methods such as microinjection, electroporation, liposomal 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人锌指蛋白8.8(Science,1984;224:1431)。 By conventional recombinant DNA techniques, the use of polynucleotide sequences of the present invention may be used to express or produce recombinant human zinc finger protein 8.8 (Science, 1984; 224: 1431). 一般来说有以下步骤:(1).用本发明的编码人人锌指蛋白8.8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;(2).在合适的培养基中培养宿主细胞;(3).从培养基或细胞中分离、纯化蛋白质。 In general the following steps: (1) encoding all the zinc present invention refers to a polynucleotide (or variant) 8.8 protein or a suitable transformed or transduced host with a recombinant expression vector comprising the polynucleotide. cells; (2) culturing the host cell in an appropriate medium; (3) isolated from the medium or the cells, the purified protein.

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。 In step (2), depending on the host cell used, the culture medium used may be selected from various conventional culture media. 在适于宿主细胞生长的条件下进行培养。 Cultured under conditions suitable for growing the host cells. 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。 When the host cells are grown to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells 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。 In step (3), the recombinant polypeptide may be coated in a cell, or expressed on the cell membrane, or secreted outside the cell. 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。 If desired, utilizing the physical, chemical and other characteristics of the isolated and purified by various separation methods of recombinant protein. 这些方法是本领域技术人员所熟知的。 These methods are well known to the skilled person. 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。 These methods include, but are not limited to: conventional renaturation treatment, protein precipitant (salting out method), centrifugation, osmosis bacteria, ultrasound treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion binding exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and 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。 Polypeptide of the present invention and antagonists, agonists and inhibitors of the polypeptide can be directly used for treatment of diseases, e.g., to treat cancer, adrenal insufficiency, skin diseases, various types of inflammation, HIV infection, and autoimmune diseases.

人C2H2型锌指蛋白是一种转录调控因子,它与基因的转录活化及抑制有关。 Human C2H2 zinc finger protein is a transcriptional regulator, which activate gene transcription and inhibition. 在体内其表达异常可引起基因的转录调控发生紊乱,导致突变蛋白质的产生,进而导致相关疾病的发生。 Expression in vivo may cause abnormal transcriptional regulation of gene disorder, resulting mutant protein, leading to the occurrence of related diseases.

本发明的多肽的表达谱与人C2H2型锌指蛋白的表达谱相一致,两者具有相似的生物学功能。 Expression profile with human type C2H2 zinc finger polypeptide of the present invention protein expression profiles consistent both have similar biological functions. 本发明的多肽在体内是一种转录调控因子,它与基因的转录活化及抑制有关。 Polypeptide of the invention in vivo is a transcriptional regulator, which activate gene transcription and inhibition. 其表达异常可引起基因的转录调控发生紊乱,导致突变蛋白质的产生,进而导致胚胎发育畸形、肿瘤、蛋白质代谢紊乱性疾病的发生,这些疾病包括但不限于:常见胚胎发育畸形一.颜面、颈和四肢的常见畸形: Expression abnormalities can cause transcriptional regulation of gene disturbed, resulting mutant protein, leading to embryonic malformations, tumors, protein metabolism disorders. These diseases include, but are not limited to: common embryonic malformations a face, neck. and common deformity of the limbs:

1.唇裂(最常见,可伴有牙槽突裂和腭裂),腭裂,面斜裂,颈囊,颈瘘等;2.四肢的常见畸形:1)肢体缺如:横向缺如(先天性短肢):无臂,无前臂,无手,无指,无腿,无趾等;纵向缺如:上肢桡/尺侧缺如,下肢胫/腓侧缺如等;海豹样手/足畸形等;2)肢体分化障碍:某块肌肉或肌群缺如,关节发育不良,骨畸形,骨融合,多指(趾),并指(趾)畸形,马碲内翻足等;二.消化系统的常见畸形:甲状舌管囊肿,消化管闭锁或狭窄,回肠憩室,脐瘘,先天性脐疝,先天性无神经节性巨结肠,不通肛,肠绊转位异常,胆管闭锁,环状胰等;三.呼吸系统的常见畸形:喉气管狭窄/闭锁,气管食管瘘,透明膜病,单侧肺不发生,异位肺叶,先天性肺囊肿,肺膨胀不全等;四.泌尿系统的常见畸形:多囊肾,异位肾,马碲肾,双输尿管,脐尿瘘,膀胱外翻等;五.生殖 1. lip (the most common, and may be associated with alveolar cleft palate), palate, surface oblique fissure, bladder neck, cervical fistula; 2 common limb abnormalities: 1) absent limb: absence of lateral (congenital short limb): None arm, forearm no, no hand, no means, no legs, no other toes; longitudinal absent: upper radial / ulnar absent, tibial / absence fibular like; comp seals hand / foot deformities and the like; 2) physical differentiation disorder: an absence of muscle or muscle group, joint dysplasia, bone deformities, bone fusion, multiple fingers (toes), and (toe) deformity, varus the horseshoe; two digested common system abnormalities: thyroglossal cyst, digestive tract atresia or stenosis, ileum diverticulitis, umbilical fistula, congenital umbilical hernia, congenital aganglionic megacolon, anal barrier, abnormal intestinal translocation trip, biliary atresia, cyclic pancreas; three common abnormal respiratory system: laryngotracheal stenosis / atresia, tracheoesophageal fistula, hyaline membrane disease, unilateral lung occurs, ectopic lobe, congenital pulmonary cyst, atelectasis; Fourth urinary system. common malformations: polycystic kidney disease, ectopic kidney, horseshoe kidney, ureter, urinary fistula umbilical, bladder exstrophy and so on; five reproduction. 统的常见畸形:隐睾,先天性腹股沟疝,双子宫,阴道闭锁,尿道下裂,真/假两性畸形,睾丸女性化综合征等;六.心血管系统的常见畸形:房间隔缺损,室间隔缺损,动脉干分隔异常(主动脉和肺动脉错位、主动脉或肺动脉狭窄),动脉导管未闭等;七.神经系统的常见畸形:神经管缺陷(无脑畸形、脊髓裂、脊髓脊膜膨出、积水性脑膜脑膨出),脑内/外脑积水等;八.眼、耳的常见畸形:虹膜缺损,瞳孔膜存留,先天性白内障,先天性青光眼,独/无/小眼畸形,先天性耳聋,耳廓畸形等;各种组织常见肿瘤:一.上皮组织:乳头状瘤,鳞状细胞癌【皮肤、鼻咽、喉、宫颈】,腺瘤(癌)【乳腺、甲状腺】,粘/浆液性囊腺瘤(癌)【卵巢】,基底细胞癌【头面部皮肤】,(恶性)多型性腺瘤【延腺】,乳头状瘤、移行上皮癌【膀胱、肾盂】等;二.间叶组织:纤维 Common conventional malformations: cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, true / pseudohermaphroditism, testicular feminization syndrome; six common abnormalities of the cardiovascular system: atrial septal defects, ventricular septal defect, truncus arteriosus partition abnormality (displacement aorta and pulmonary artery, aortic or pulmonary stenosis), patent ductus arteriosus, and the like; seven common abnormalities of the nervous system: neural tube defects (anencephaly, spinal bifida, myelomeningocele expansion out of the water meningoencephalocele), brain / outer hydrocephalus; eight common deformity eye, ear: coloboma, pupillary membrane retention, congenital cataracts, congenital glaucoma, only / no / ommatidium malformations, congenital deafness, ear deformities; organizations common tumors: a epithelium: papilloma, squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (cancer) [breast, thyroid ], viscous / serous cystadenoma (carcinoma) ovarian [], basal cell carcinoma, head and facial skin [], (malignant) polymorphism extension gland adenoma [], papilloma, transitional cell carcinoma of the bladder [,], and other renal pelvis ; two leaf tissue: fibrous (肉)瘤【四肢】,(恶性)纤维组织细胞瘤【四肢】,脂肪(肉)瘤【皮下组织、下肢、腹膜后】,平滑肌(肉)瘤【子宫和胃肠】,横纹肌(肉)瘤【头颈、生殖泌尿道、四肢】,血管(肉)瘤、淋巴管(肉)瘤【皮肤、皮下组织、舌、唇】,骨(肉)瘤【颅骨、长骨】,(恶)性巨细胞瘤【股/胫/肱骨上端】,软骨(肉)瘤【手足短骨、盆/肋/股/肱/肩胛骨】,滑膜(肉)瘤【膝/踝/腕/肩/肘关节附近】,(恶)性间皮瘤【胸/腹膜】等;三.淋巴造血组织:恶性淋巴瘤【颈部、纵隔、肠系膜和腹膜后淋巴结】,各种白血病【淋巴造血组织】,多发性骨髓瘤【椎/胸/肋/颅骨和长骨】等;四.神经组织:神经纤维(肉)瘤【全身皮神经/深部神经及内脏】,(恶性)神经鞘瘤【头、颈、四肢等处神经】,(恶性)胶质细胞瘤【大脑】,髓母细胞瘤【小脑】,(恶性)脑膜瘤【脑膜】,节神经细胞瘤/ (Meat) tumors [limbs], (malignant) fibrous histiocytoma] [limbs, fat (meat) [subcutaneous tumor, lower extremity, retroperitoneal], smooth muscle (meat) uterine and gastrointestinal tumors [], striated muscle (meat) tumors [neck, genitourinary tract, limbs], vascular (meat) tumors, lymphatic (meat) tumors [skin, subcutaneous tissue, tongue, lips], bone (meat) tumors [skull, long bones], (evil) of giant cell tumors [tibiofemoral / humerus /], cartilage (meat) tumors [short hand and foot bones, pots / rib / share / humerus / scapula], synovial (meat) near the tumor [knee / ankle / wrist / shoulder / elbow .], (evil) mesothelioma [chest / ip]; three hematopoietic and lymphoid tissues: neck malignant lymphoma, mediastinal, mesenteric and retroperitoneal lymph node], various hematopoietic tissues lymphoid leukemia [], multiple myeloma tumor [vertebral / chest / rib / skull and long bones]; Fourth nerve tissue: nerve fibers (meat) tumors [systemically nerve / deep nerves and internal organs], (malignant) schwannoma [head, neck, limbs, etc. nerve], (malignant) astrocytoma [brain], cerebellar medulloblastoma [], (malignant) meningioma [meninges], section neuroblastoma / 经母细胞瘤【纵隔和腹膜后/肾上腺髓质】等;五.其他肿瘤:黑痣,恶性黑色素瘤【皮肤、粘膜】,(恶性)葡萄胎,绒毛膜上皮癌【子宫】,(恶性)支持细胞、间质细胞瘤,(恶性)颗粒细胞瘤【卵巢、睾丸】,精原细胞瘤【睾丸】,无性细胞瘤【卵巢】,胚胎性癌【睾丸、卵巢】,(恶性)畸胎瘤【卵巢、睾丸、纵隔和骶尾部】等;蛋白质代谢紊乱相关疾病蛋白质代谢紊乱可影响蛋白质下列主要生理功能,进而导致相关疾病的发生:一.提供机体能量,维持组织生长、更新和修补:肌肉萎缩,四肢软弱,身体消瘦,严重者可呈“恶液质”表现;二.产生一些生理活性物质,如激素、抗体、胺类等:1.蛋白质肽类激素功能紊乱可导致如下疾病发生:1)胰岛素和胰高血糖素:糖尿病,低血糖症等;2)下丘脑和垂体激素:巨人症,侏儒症,肢端肥大症,皮质醇增多 [Blastoma through mediastinal and retroperitoneal / adrenal medulla, etc.]; the other five tumors: mole, [malignant melanoma of the skin, mucous membranes], (malignant) mole, choriocarcinoma [uterine], (malignant). Sertoli cell, Leydig cell tumors, (malignant) [granulosa cell tumor of the ovary, testis], testicular seminoma [], ovarian dysgerminoma [], embryonal carcinoma [testes, ovary], (malignant) teratomas [ovary, testis, mediastinum, etc.], and sacrococcygeal; protein metabolism disorder diseases may affect protein metabolism following main physiological function of the protein, leading to the occurrence of a disease associated with: a body providing energy to maintain the tissue growth, repair and updating: muscle. atrophy, limb weakness, body weight loss, severe cases may be in the "cachexia" performance; two produce some physiologically active substances, such as hormones, antibodies, amines and the like: a protein peptide hormone dysfunction can cause the following diseases: 1) insulin and glucagon: diabetes, hypoglycemia; 2) the hypothalamus and pituitary hormones: gigantism, dwarfism, acromegaly, Cushing 症(柯兴综合症),原发性性醛固酮增多症,继发慢性肾上腺皮质功能减退,甲状腺机能亢进症,甲状腺机能减退症(呆小病,幼年型甲减,成年型甲减),男/女不育症,月经失调(功能性子宫出血、闭经、多囊卵巢综合症、经前期紧张综合症、更年期综合症),性发育障碍,尿崩症、抗利尿激素分泌不当综合症,泌乳异常等;3)甲状旁腺素:甲状旁腺机能亢进症,甲状旁腺机能减退症等; Disease (Cushing's syndrome), primary aldosteronism, adrenal secondary to chronic hypothyroidism, hyperthyroidism, hypothyroidism (cretinism, juvenile hypothyroidism, adult hypothyroidism), M / female infertility, menstrual disorders (dysfunctional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, menopausal syndrome), developmental disorders, diabetes insipidus, syndrome of inappropriate secretion of antidiuretic hormone, lactation abnormal; 3) parathyroid hormone: hyperparathyroidism, hypoparathyroidism psychosis;

4)胃肠道激素:消化性溃疡,慢性消化不良,慢性胃炎等;2.胺类物质代谢紊乱可导致如下疾病发生:心律失常,休克,精神错乱,癫痫,舞蹈病,肝性脑病(去甲肾上腺素、γ-氨基丁酸、5-羟色胺、谷氨酰胺),晕动病,I-型变态反应性疾病(荨麻疹、枯草热、过敏性鼻炎、皮肤过敏),消化性溃疡(组胺),高胆固醇血症(牛磺酸),肿瘤(多胺)等;3.抗体缺陷易发生各种感染:败血症,化脓性脑膜炎,肺炎,气管炎,中耳炎,脓皮病等;三.某些蛋白质的特殊生理作用:各种血红蛋白病(贫血、黄疸、组织缺氧致有机酸血症),各种凝血因子缺乏症(出血),肌痉挛,肌强制、肌麻痹(肌动蛋白)、高脂蛋白血症等;综合上述,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多种疾病的治疗,例如胚胎发育畸形、肿瘤、蛋白质代谢紊乱性疾病等。 4) Gastrointestinal Hormones: peptic ulcer, chronic indigestion, chronic gastritis; 2 amines metabolic disorders can cause the following diseases: arrhythmia, shock, psychosis, epilepsy, chorea, hepatic encephalopathy (to norepinephrine, [gamma] -aminobutyric acid, serotonin, glutamine), motion sickness, I- type allergic diseases (urticaria, hay fever, allergic rhinitis, skin allergies), peptic ulcer (group amine), hypercholesterolemia (taurine), tumor (polyamines); 3 antibody deficiency prone to various infections: sepsis, purulent meningitis, pneumonia, bronchitis, otitis media, pyoderma; three special physiological role of certain proteins: various hemoglobinopathies (anemia, jaundice, hypoxia-induced hyperlipidemia acids), various coagulation factor deficiency (bleeding), muscle spasms, muscle force, muscle paralysis (actin ), hyperlipoproteinemia and the like; in summary, the present invention and polypeptide antagonists of the polypeptide, agonists and inhibitors may be used directly for treatment of various diseases, such as embryonic malformations, tumors, protein metabolism disorders Wait.

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)人锌指蛋白8.8的药剂的方法。 The present invention also provides methods of screening compounds to identify enhance (agonist) or repression (antagonists) agents of human zinc finger protein 8.8. 激动剂提高人锌指蛋白8.8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。 Agonists improve the human zinc finger protein 8.8 stimulate cell proliferation and other biological functions, while preventing the antagonist and treatment of disorders related to excessive cell proliferation such as various cancers. 例如,能在药物的存在下,将哺乳动物细胞或表达人锌指蛋白8.8的膜制剂与标记的人锌指蛋白8.8-起培养。 For example, the drug can be in the presence of the mammalian cell or membrane preparation expressing the human zinc finger protein labeled with 8.8 of human zinc finger protein from the culture 8.8. 然后测定药物提高或阻遏此相互作用的能力。 Drug is then determined or improve the ability of this repressor interactions.

人锌指蛋白8.8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。 8.8 of human zinc finger protein selected antibody antagonists include the compound, receptor and deletion analogs thereof and the like. 人锌指蛋白8.8的拮抗剂可以与人锌指蛋白8.8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。 8.8 human zinc finger protein antagonist may bind human zinc finger protein 8.8 and eliminate its function, or inhibiting production of the polypeptide or binding to the active site of the polypeptide so that the polypeptide can not exert a biological function.

在筛选作为拮抗剂的化合物时,可以将人锌指蛋白8.8加入生物分析测定中,通过测定化合物对人锌指蛋白8.8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。 At screening as an antagonist compound may be added to human zinc finger protein 8.8 Determination organism, human zinc finger protein interaction effect between 8.8 and its receptors by determining a compound to determine whether the compound is an antagonist. 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。 Same manner as for screening compounds may act as antagonists selected receptor and deletion analogs thereof. 能与人锌指蛋白8.8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。 Zinc finger polypeptide can be human binding molecule may be a protein binding to a 8.8 random peptide library composed of a solid phase by screening all possible combinations of amino acids is obtained. 筛选时,一般应对人锌指蛋白8.8分子进行标记。 Screening, people generally respond zinc finger protein molecules labeled 8.8.

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。 The present invention provides a polypeptide, and fragments, derivatives, analogs thereof, or cells as an antigen in producing the antibody. 这些抗体可以是多克隆抗体或单克隆抗体。 These antibodies can be polyclonal or monoclonal antibodies. 本发明还提供了针对人锌指蛋白8.8抗原决定簇的抗体。 The present invention also provides for human zinc finger protein antigenic determinant of antibody 8.8. 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。 These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and Fab expression library fragments generated.

多克隆抗体的生产可用人锌指蛋白8.8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。 Production of polyclonal antibodies available human zinc finger protein 8.8 direct injection immunizing an animal (e.g., rabbit, mouse, rat, etc.) obtained by the method, various adjuvants may be used to enhance the immune response, including but not limited to Freund's adjuvant . 制备人锌指蛋白8.8的单克隆抗体的技术包括但不限于杂交瘤技术(Kohler andMilstein.Nature,1975,256:495-497),三瘤技术,人B-细胞杂交瘤技术,EBV-杂交瘤技术等。 The preparation of human monoclonal antibody technology zinc finger proteins include, but are not limited to 8.8 hybridoma technique (Kohler andMilstein.Nature, 1975,256: 495-497), three hybridoma technique, the human B- cell hybridoma technique, the EBV-hybridoma technology. 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)。 The chimeric antibody variable region and a human constant region of non-human origin in combination produce the available prior art (Morrison et al, PNAS, 1985,81: 6851). 而已有的生产单链抗体的技术(USPat No.4946778)也可用于生产抗人锌指蛋白8.8的单链抗体。 Only some of the technical production of single chain antibodies (USPat No.4946778) may also be used to produce single-chain anti-human antibody zinc finger protein 8.8.

抗人锌指蛋白8.8的抗体可用于免疫组织化学技术中,检测活检标本中的人锌指蛋白8.8。 8.8 human anti-zinc finger protein antibodies can be used in immunohistochemical techniques, biopsy specimens detect human zinc finger protein 8.8.

与人锌指蛋白8.8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。 Monoclonal antibodies with human zinc finger protein binding 8.8 radiolabels can also be used, it can be injected into the body to track their location and distribution. 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。 Whether this radiolabeled antibody may be used as a non-invasive diagnostic methods for determining and locating tumor cells metastasis.

抗体还可用于设计针对体内某一特殊部位的免疫毒素。 Antibodies can also be used to design immunotoxins for a particular site in the body. 如人锌指蛋白8.8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。 The zinc finger protein 8.8 human high affinity monoclonal antibodies may be combined with a bacterial or plant toxin (such as diphtheria toxin, ricin, beans bases, etc.) covalently. 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锌指蛋白8.8阳性的细胞。 One common method is to use mercapto-crosslinkers such as SPDP, amino antibodies attack by disulfide exchange, the toxin bound to the antibody, such hybrid antibodies may be used to kill human zinc finger protein 8.8 positive cells.

本发明中的抗体可用于治疗或预防与人锌指蛋白8.8相关的疾病。 The antibodies of the invention may be used to treat or prevent human zinc finger protein related diseases 8.8. 给予适当剂量的抗体可以刺激或阻断人锌指蛋白8.8的产生或活性。 Antibodies can be administered the appropriate dose of stimulating or blocking the production or activity of human zinc finger protein 8.8.

本发明还涉及定量和定位检测人锌指蛋白8.8水平的诊断试验方法。 The present invention further relates to the location detection and quantitative diagnostic tests human zinc finger protein 8.8 level. 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。 These tests are well known in the art, and include FISH assay and radioimmunoassay. 试验中所检测的人锌指蛋白8.8水平,可以用作解释人锌指蛋白8.8在各种疾病中的重要性和用于诊断人锌指蛋白8.8起作用的疾病。 The assay detected 8.8 human zinc finger protein levels may serve to explain the importance of the human zinc finger protein 8.8 in various diseases and for the diagnosis of human zinc finger protein 8.8 acting diseases.

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。 Polypeptides of the invention are also useful as peptide mapping, e.g., polypeptides can be physical, chemical or specific cleavage enzyme, and a one-dimensional gel electrophoresis or two-dimensional or three-dimensional, more preferably subjected to mass spectrometry.

编码人锌指蛋白8.8的多核苷酸也可用于多种治疗目的。 Polynucleotides encoding zinc finger proteins can also be 8.8 for various therapeutic purposes. 基因治疗技术可用于治疗由于人锌指蛋白8.8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。 Gene therapy techniques may be useful in the treatment since the human zinc finger protein 8.8 no expression or abnormal / non-activity-induced expression of cell proliferation, development or metabolic abnormalities. 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锌指蛋白8.8,以抑制内源性的人锌指蛋白8.8活性。 Recombinant human gene therapy vectors (e.g., viral vectors) can be designed to express variant zinc finger protein 8.8, inhibition of endogenous human zinc finger protein activity 8.8. 例如,一种变异的人锌指蛋白8.8可以是缩短的、缺失了信号传导功能域的人锌指蛋白8.8,虽可与下游的底物结合,但缺乏信号传导活性。 For example, a variant of human zinc finger protein 8.8 may be shortened, missing signal transduction domain of human zinc finger protein 8.8, although the substrate may be combined with the downstream, but lack signal transduction activity. 因此重组的基因治疗载体可用于治疗人锌指蛋白8.8表达或活性异常所致的疾病。 Thus recombinant gene therapy vectors may be used for the treatment of human zinc finger protein 8.8 caused by abnormal expression or activity of disease. 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锌指蛋白8.8的多核苷酸转移至细胞内。 Expression vectors derived from viruses such as retroviruses, adenoviruses, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used for encoding the zinc finger protein polynucleotide transferred into the cells of 8.8. 构建携带编码人锌指蛋白8.8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.)。 Construction method of carrying the zinc finger protein encoding polynucleotide 8.8 recombinant viral vectors can be found in existing literature (Sambrook, et al.). 另外重组编码人锌指蛋白8.8的多核苷酸可包装到脂质体中转移至细胞内。 Further recombinant polynucleotide encoding a zinc finger protein 8.8 can be packaged into liposomes 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。 Introduction of polynucleotides into a cell or tissue in a method comprising: the polynucleotide is injected directly into the tissue in vivo; or in vitro by a vector (e.g., a virus, phage or plasmid, etc.) first polynucleotide into a cell, then the cell transplantation into the body and so on.

抑制人锌指蛋白8.8mRNA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。 Inhibition of human zinc finger protein 8.8mRNA oligonucleotides (including antisense RNA and DNA) and ribozymes are also within the scope of the present invention. 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。 A ribozyme capable of specific RNA molecules like enzyme-specific RNA, which mechanism of action is specific hybridization of the ribozyme molecule to complementary target RNA endonuclease action is performed. 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。 DNA and antisense RNA and ribozyme RNA or DNA using any conventional synthetic techniques available, such as solid-phase synthesis of phosphate chemical synthesis of oligonucleotides has been widely used in the art. 反义RNA分子可通过编码该RNA的DNA序列在体外或体内转录获得。 Antisense RNA molecule may be encoded by a DNA sequence of the RNA in vitro or in vivo transcription. 这种DNA序列已整合到载体的RNA聚合酶启动子的下游。 This DNA sequence is integrated into the vector downstream of the promoter of RNA polymerase. 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。 In order to increase the stability of the nucleic acid molecule, using a variety of methods for their modification, such as increasing the connected applications or phosphoric acid thioester bonds peptide bonds rather than phosphodiester bonds between the sequence length, on both sides of the ribonucleoside.

编码人锌指蛋白8.8的多核苷酸可用于与人锌指蛋白8.8的相关疾病的诊断。 A polynucleotide encoding a human zinc finger protein 8.8 may be used in diagnosis of human disease associated zinc finger protein 8.8. 编码人锌指蛋白8.8的多核苷酸可用于检测人锌指蛋白8.8的表达与否或在疾病状态下人锌指蛋白8.8的异常表达。 A polynucleotide encoding a human zinc finger protein 8.8 may be used to detect the expression of human zinc finger protein 8.8 or absence or abnormal expression of the zinc finger protein 8.8 Human disease state. 如编码人锌指蛋白8.8的DNA序列可用于对活检标本进行杂交以判断人锌指蛋白8.8的表达状况。 The DNA sequence encoding human zinc finger protein 8.8 biopsy specimens may be used to hybridize to determine the expression of human zinc finger protein 8.8. 杂交技术包括Soutbern印迹法,Northern印迹法、原位杂交等。 Hybridization techniques include Soutbern blotting, Northern blotting, and in situ hybridization. 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。 These technologies mature technology methods are disclosed, related kits can be obtained from commercial sources. 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。 Portion of the polynucleotide or all of the present invention may be used as probes are immobilized on a microarray (the Microarray) or DNA chip (also known as "gene chips"), the analysis of differences in tissue for gene diagnosis and gene expression analysis. 用人锌指蛋白8.8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锌指蛋白8.8的转录产物。 8.8 human zinc finger protein specific primers RNA- polymerase chain reaction (RT-PCR) amplification may be detected in vitro human zinc finger protein transcription product 8.8.

检测人锌指蛋白8.8基因的突变也可用于诊断人锌指蛋白8.8相关的疾病。 Detecting a human mutein 8.8 zinc finger gene may also be used to diagnose human zinc finger protein related diseases 8.8. 人锌指蛋白8.8突变的形式包括与正常野生型人锌指蛋白8.8DNA序列相比的点突变、易位、缺失、重组和其它任何异常等。 8.8 human zinc finger protein comprises a mutated form of the normal human wild-type zinc finger protein 8.8DNA point mutation compared sequences, translocation, deletion, rearrangement, and any other abnormalities. 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。 Available conventional techniques such as Southern blotting, DNA sequencing, PCR and in situ hybridization detection of mutations. 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。 Further, mutations may affect protein expression, thus by Northern blotting, Western blotting method can indirectly determine presence or absence of gene mutations.

本发明的序列对染色体鉴定也是有价值的。 Sequences of the invention are also valuable for chromosome identification. 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。 The sequence will specifically chromosome specific location and can hybridize therewith and a bar for people. 目前,需要鉴定染色体上的各基因的具体位点。 At present, the need to identify specific sites for each gene on the chromosome. 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。 Now, based on only a few actual sequence data (repeat polymorphisms) are useful for chromosome marking marking chromosomal location. 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。 According to the present invention, to those sequences associated with disease-related genes, which is an important first step of these DNA sequences located on 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。 Briefly, cDNA was prepared according to the PCR primers (preferably 15 to 35 bp), the sequence may be located on the chromosome. 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。 Then, these primers were used for PCR screening of human chromosomes containing the pieces of somatic cell hybrid cell. 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。 Only those hybrid cell containing the human gene corresponding to the primer will produce an amplified fragment.

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。 PCR method targeting somatic cell hybrid cell, target DNA is a quick way of specific chromosomes. 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。 The present invention oligonucleotide primers, by similar methods, may be implemented using a set of sub-positioning fragments from specific chromosomes or large genomic clones. 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。 Other similar strategy can be used include chromosomal location in situ hybridization, prescreening and hybridization with a preselected flow-sorted chromosomal markers to construct chromosome-specific cDNA libraries.

将cDNA克隆与中期染色体进行荧光原位杂交(FISH),可以在一个步骤中精确地进行染色体定位。 The cDNA clone with metaphase chromosome fluorescence in situ hybridization (FISH), chromosome can be accurately positioned in one step. 此技术的综述,参见Verma等,Human Chromosomes:a Manualof Basic Techniques,Pergamon Press,New York(1988)。 Review of this technique, see Verma et al., Human Chromosomes: a Manualof 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。 Once the sequence is positioned to the precise chromosomal location, the physical position of the sequence on the chromosome can be associated with genetic map data. 这些数据可见于例如,V.Mckusick,MendelianInheritance in Man(可通过与Johns Hopkins UniVersity Welch Medical Library联机获得)。 These data can be found in e.g., V.Mckusick, MendelianInheritance in Man (available through Johns Hopkins UniVersity Welch Medical Library available online). 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。 Then identified through linkage analysis, to identify genes and has been positioned on the relationship between the disease chromosomal region.

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。 Subsequently, the cDNA sequence is necessary to measure differences between diseased and non-diseased individuals, or genome. 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。 If observed in some or all of the affected individuals to a mutation, but the mutation is not in any normal individuals was observed, then the mutation is likely to be the cause of the disease. 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。 Comparison of diseased and non-diseased individuals generally involves first looking for structural chromosome changes, as can be seen from the chromosomal level or by PCR cDNA sequence based on a detectable deletions or translocations. 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)。 The resolution of the current physical mapping and gene mapping techniques, cDNA is precisely positioned to the chromosomal region associated with the disease, and may be one of between 50-500 potential causative genes (assuming 1 megabase mapping resolution 20kb per capacity corresponding 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。 Polypeptides of the invention may be, and the polynucleotide, agonist, antagonist, and a suitable carrier composition mimetic inhibitor drug use. 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。 The carrier may be water, dextrose, ethanol, salts, buffers, glycerin, and combinations thereof. 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。 The composition comprises a safe and effective amount of the polypeptide or antagonist and pharmaceutical excipients and carriers do not affect the effect. 这些组合物可以作为药物用于疾病治疗。 These compositions can be used as a medicament 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。 The present invention further provides a container containing one or more kits or kits, containing one or more containers of the present invention, pharmaceutical composition ingredients. 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。 Together with these containers, there can be indicative prompted by the manufacture, use or sale of government regulatory agencies drugs or biological products are given, the prompt reflects the production, use or sale of government agencies that license applied on the human body. 此外,本发明的多肽可以与其它的治疗化合物结合使用。 In addition, polypeptides of the invention may be used in conjunction 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。 The pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. 人锌指蛋白8.8以有效地治疗和/或预防具体的适应症的量来给药。 8.8 at human zinc finger and / or prevention of an amount effective to treat a particular indication administered protein. 施用于患者的人锌指蛋白8.8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。 Who administered to a patient an amount of a zinc finger protein 8.8 dosage range and will depend on many factors, such as health condition and the doctor's diagnosis mode of administration, the subject to be determined.

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 serve to illustrate specific embodiments of the present invention and are not intended to limit the claims as defined by the scope of the invention.

图1是本发明人锌指蛋白8.8和人锌指蛋白的基因芯片表达谱比较图。 The present invention, FIG. 1 is a zinc finger protein 8.8 gene and human zinc finger protein microarray expression of comparison of FIG. 上图是人锌指蛋白8.8的表达谱折方图,下方序列是人锌指蛋白的表达谱折方图。 The figure is a human zinc finger protein expression profile of 8.8 fold Square sequence is under the expression profile of human zinc finger protein folding side of FIG. 其中,1-胎脑、2-膀胱粘膜、3-PMA+的Ecv304细胞株、4-LPS+的Ecv304细胞株胸腺、5-正常成纤维细胞1024NC、6-Fibroblast,生长因子刺激,1024NT、7-疤痕成fc生长因子刺激,1013HT、8-疤痕成fc未用生长因子刺激,1013HC、9-膀胱癌建株细胞EJ、10-膀胱癌旁、11-膀胱癌、12-肝癌、13-肝癌细胞株、14-胎皮、15-脾脏、16-前列腺癌、17-空肠腺癌、18贲门癌。 Wherein, 1 fetal brain, bladder mucosa 2-, 3-PMA + of Ecv304 cell line, 4-LPS + Ecv304 the thymus cell line, 5-normal fibroblasts 1024NC, 6-Fibroblast, growth factor stimulation, 1024NT, 7- scars fc to growth factor stimulation, 1013HT, 8- to fc scars not stimulated with growth factors, 1013HC, 9- built bladder cancer cell lines EJ, bladder carcinoma adjacent 10-, 11- bladder cancer, hepatoma 12-, 13- hepatoma cell line , 14 fetal skin, spleen 15, 16 prostate cancer, 17 jejunum cancer, 18 gastric cardia.

图2为分离的人锌指蛋白8.8的聚丙烯酰胺凝胶电泳图(SDS-PAGE)。 Figure 2 is an isolated human zinc finger protein polyacrylamide gel electrophoresis is 8.8 (SDS-PAGE). 9kDa为蛋白质的分子量。 9kDa molecular weight proteins. 箭头所指为分离出的蛋白条带。 Arrows indicate protein bands separated.

下面结合具体实施例,进一步阐述本发明。 The following embodiments with reference to specific embodiments, further illustrate the present invention. 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。 It should be understood that these embodiments are illustrative only and the present invention is not intended to limit the scope of the invention. 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:ColdSpring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。 Experimental methods without specific conditions in the examples below, generally in accordance with conventional conditions as described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989) in the condition, or instructed by the manufacturers It recommended conditions. 实施例1:人锌指蛋白8.8的克隆用异硫氰酸胍/酚/氯仿一步法提取人胎脑总RNA。 Example 1: Human zinc finger clones Total RNA from human fetal brain protein extract 8.8 guanidinium isothiocyanate / phenol / chloroform step process. 用Quik mRNA Isolation Kit(Qiegene公司产品)从总RNA中分离poly(A)mRNA。 Quik mRNA Isolation Kit (Qiegene Products) isolating poly (A) mRNA from total RNA using. 2ug poly(A)mRNA经逆转录形成cDNA。 2ug poly (A) mRNA is formed by reverse transcription 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DH5α,细菌形成cDNA文库。 With the Smart cDNA cloning kit (available from Clontech) cDNA fragment was cloned into the pBSK (+) multiple cloning site of the vector (Clontech Co.), and transformed DH5 [alpha], the bacteria form a cDNA library. 用Dyeterminate 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。 And ABI 377 automated sequencer (Perkin-Elmer Company) determining the sequence of all cloned 5 'and 3' ends with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer Co.). 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0132g08的cDNA序列为新的DNA。 The determined cDNA sequence and DNA sequence existing public databases (Genebank accession) were compared, found a clone cDNA sequence 0132g08 new 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。 Determination of the bidirectional clones contained inserted cDNA fragments by synthesis of a series of primers. 结果表明,0132g08克隆所含的全长cDNA为716bp(如Seq ID NO:1所示),从第80bp至464bp有一个242bp的开放阅读框架(ORF),编码一个新的蛋白质(如Seq IDNO:2所示)。 The results showed that clones contained full-length cDNA 0132g08 of 716bp (eg Seq ID NO: 1 below), a 242bp open reading frame (ORF) from 80bp to 464bp, encoding a novel protein (e.g. Seq IDNO: FIG 2). 我们将此克隆命名为pBS-0132g08,编码的蛋白质命名为人锌指蛋白8.8。 We named this clone pBS-0132g08, encoding a protein named human zinc finger protein 8.8. 实施例2:用RT-PCR方法克隆编码人锌指蛋白8.8的基因用胎脑细胞总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:Primer1:5'-GAAGCACTTTCTTAAGACTGACCT-3'(SEQ ID NO:3)Primer2:5'-TACAATAAATCTGATTGATTTTTA-3'(SEQ ID NO:4)Primer1为位于SEQ ID NO:1的5'端的第1bp开始的正向序列;Primer2为SEQ ID NO:1的中的3'端反向序列。 Example 2: RT-PCR to clone encoding the zinc finger protein 8.8 using fetal brain cells total RNA as a template and oligo-dT as a primer for reverse transcription reaction synthesizing the cDNA, after purification kit Qiagene by using the following primers for PCR amplification: Primer1: 5'-GAAGCACTTTCTTAAGACTGACCT-3 '(SEQ ID NO: 3) Primer2: 5'-TACAATAAATCTGATTGATTTTTA-3' (SEQ ID NO: 4) Primer1 located SEQ ID NO: 5 'end of the 1 1bp start of forward sequence; Primer2 of SEQ ID NO: 1, 3 'end of the reverse sequence.

扩增反应的条件:在50μl的反应体积中含有50mmol/L KCl,10mmol/LTris-Cl,(pH8.5),1.5mmol/L MgCl2,200μmol/L dNTP,10pmol引物,1U的Taq DNA聚合酶(Clontech公司产品)。 Amplification reaction conditions: reaction volume containing 50μl of 50mmol / L KCl, 10mmol / LTris-Cl, (pH8.5), 1.5mmol / L MgCl2,200μmol / L dNTP, 10pmol primer, 1U Taq DNA polymerase (Clontech company's products). 在PE9600型DNA热循环仪(Perkin-Elmer公司)上按下列条件反应25个周期:94℃ 30sec;55℃ 30sec;72℃ 2min。 On PE9600 DNA Thermal Cycler (Perkin-Elmer Co.) by the reaction of 25 cycles of the following conditions: 94 ℃ 30sec; 55 ℃ 30sec; 72 ℃ 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。 In RT-PCR β-actin provided simultaneously as a positive control and a negative control as blank templates. 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)。 Amplified product was purified using a QIAGEN kit, connected to the pCR vector (Invitrogen Corporation) using TA cloning kit. DNA序列分析结果表明PCR产物的DNA序列与SEQ ID NO:1所示的1-716bp完全相同。 DNA sequence analysis showed that the PCR product DNA sequence SEQ ID NO: 1-716bp exactly shown. 实施例3:Northern印迹法分析人锌指蛋白8.8基因的表达:用一步法提取总RNA[Anal.Biochem 1987,162,156-159]。 Example 3: Northern blotting analysis of expression of human zinc finger protein gene 8.8: Extraction of total RNA [Anal.Biochem 1987,162,156-159] by one step. 该法包括酸性硫氰酸胍苯酚-氯仿抽提。 Guanidinium thiocyanate acid which comprises phenol - chloroform extraction. 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。 I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0.2M sodium acetate (pH 4.0) of the tissue was homogenized, 1 volume of phenol and 1/5 volume of chloroform - isoamyl alcohol (49:1 ), mixed after centrifugation. 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。 Water absorbing layer was added isopropyl alcohol (0.8 vol) was added and the mixture was centrifuged precipitate RNA. 将得到的RNA沉淀用70%乙醇洗涤,干燥并溶于水中。 The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 用20μg RNA,在含20mM 3-(N-吗啉代)丙磺酸(pH7.0)-5mM乙酸钠-1mM EDTA-2.2M甲醛的1.2%琼脂糖凝胶上进行电泳。 With 20μg RNA, (N- morpholino) propanesulfonic acid (pH7.0) was subjected to electrophoresis on a 1.2% agarose gel -5mM -1mM EDTA-2.2M sodium acetate formaldehyde containing 20mM 3-. 然后转移至硝酸纤维素膜上。 Then transferred to a nitrocellulose membrane. 用α-32P dATP通过随机引物法制备32P-标记的DNA探针。 32P- labeled DNA probe prepared by random priming using Method α-32P dATP. 所用的DNA探针为图1所示的PCR扩增的人锌指蛋白8.8编码区序列(80bp至464bp)。 The DNA probe used is shown in FIG. 1 PCR amplified human zinc finger protein 8.8 coding sequence (80bp to 464bp). 将32P-标记的探针(约2×106cpm/ml)与转移了RNA的硝酸纤维素膜在一溶液中于42℃杂交过夜,该溶液包含50%甲酰胺-25mM KH2PO4(pH7.4)-5×SSC-5×Denhardt's溶液和200μg/ml鲑精DNA。 The 32P- labeled probe (approximately 2 × 106cpm / ml) and RNA was transferred to nitrocellulose membrane hybridized overnight at 42 ℃ a solution, the solution comprising 50% formamide -25mM KH2PO4 (pH7.4) - 5 × SSC-5 × Denhardt's solution and 200μg / ml salmon sperm DNA. 杂交之后,将滤膜在1×SSC-0.1%SDS中于55℃洗30min。 After hybridization, the filters were washed at 55 ℃ 30min in 1 × SSC-0.1% SDS in. 然后,用Phosphor Imager进行分析和定量。 Then, analyzed and quantified by Phosphor Imager. 实施例4:重组人锌指蛋白8.8的体外表达、分离和纯化根据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:Primer3:5'-CCCCATATGATGGATATCAGTTCTGCCTCACAT-3'(Seq ID No:5)Primer4:5'-CATGGATCCTCAGGAAACAAGGGCAGGCAGAAA-3'(Seq ID No:6)此两段引物的5'端分别含有NdeI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,NdeI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。 Example 4: Recombinant expression of human zinc finger protein 8.8 vitro, isolated and purified according to SEQ ID NO: 1 coding sequence shown in FIG. 1 and designed a pair of specific amplification primer sequences were as follows: Primer3: 5 ' -CCCCATATGATGGATATCAGTTCTGCCTCACAT-3 '(Seq ID No: 5) Primer4: 5'-CATGGATCCTCAGGAAACAAGGGCAGGCAGAAA-3': 5 'end (Seq ID No 6) this two primers each containing NdeI and BamHI restriction sites, respectively, followed by selective endonuclease site within the gene 5 'end and 3' end of the coding sequence, NdeI and BamHI restriction sites corresponding to the expression plasmid pET-28b (+) (Novagen products, Cat.No.69865.3) on . 以含有全长目的基因的pBS-0132g08质粒为模板,进行PGR反应。 In the plasmid pBS-0132g08 containing the full-length gene of interest as template PGR reactions. PCR反应条件为:总体积50μl中含pBS-0132g08质粒10pg、引物Primer-3和Primer-4分别为10pmol、Advantage polymerase Mix(Clontech公司产品)1μl。 PCR reaction conditions were: total volume of 50μl containing plasmid pBS-0132g08 10pg, primer Primer-3 and Primer-4, respectively 10 pmol,Advantage polymerase Mix (Clontech Products) 1μl. 循环参数:94℃ 20s,60℃ 30s,68℃ 2min,共25个循环。 Cycling parameters: 94 ℃ 20s, 60 ℃ 30s, 68 ℃ 2min, 25 cycles total. 用NdeI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。 PCR products and plasmids were pET-28 (+) double-digested with NdeI and a BamHI, the large fragment were recovered, and treated with T4 ligase. 连接产物转化用氯化钙法大肠杆细菌DH5α,在含卡那霉素(终浓度30μg/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。 Ligation products were transformed by the calcium chloride method bacteria Escherichia coli DH5 [alpha], the (final concentration 30μg / ml) were cultured overnight in LB plates containing kanamycin, positive clones were screened by colony PCR method, and sequenced. 挑选序列正确的阳性克隆(pET-0132g08)用氯化钙法将重组质粒转化大肠杆菌BL21(DE3)plySs(Novagen公司产品)。 Selected positive clones with the correct sequence (pET-0132g08) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) plySs (Novagen Co.). 在含卡那霉素(终浓度30μg/ml)的LB液体培养基中,宿主菌BL21(pET-0132g08)在37℃培养至对数生长期,加入IPTG至终浓度1mmol/L,继续培养5小时。 In containing kanamycin (final concentration 30μg / ml) in LB liquid medium, host strain BL21 (pET-0132g08) incubated at 37 [deg.] C to logarithmic phase, IPTG was added to a final concentration of 1mmol / L, and cultured 5 hour. 离心收集菌体,经超声波破菌,离心收集上清,用能与6个组氨酸(6His-Tag)结合的亲和层析柱His.Bind Quick Cartridge(Novagen公司产品)进行层析,得到了纯化的目的蛋白人锌指蛋白8.8。 Harvested by centrifugation, broken by ultrasonic bacteria, the supernatant was collected by centrifugation, chromatography can be combined with six histidine (6His-Tag) affinity chromatography His.Bind Quick Cartridge (Novagen Co.), to give purified human protein zinc finger protein 8.8. 经SDS-PAGE电泳,在9kDa处得到一单一的条带(图2)。 By SDS-PAGE electrophoresis and a single band (FIG. 2) at 9kDa. 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。 The strip was transferred to PVDF membrane for N- terminal amino acid sequence analysis by Edams hydrolysis, the results with N- terminal 15 amino acids of SEQ ID NO: N- 15 terminal amino acid residues identical to FIG. 2. 实施例5抗人锌指蛋白8.8抗体的产生用多肽合成仪(PE公司产品)合成下述人锌指蛋白8.8特异性的多肽:NH2-Met-Asp-Ile-Ser-Ser-Ala-Ser-His-Phe-Pro-Leu-Glu-Val-Glu-Ala-COOH(SEQ ID NO:7)。 Example 5 Anti-human zinc finger protein to produce antibodies 8.8 below was synthesized human polypeptide synthesizer (PE Corporation) 8.8 zinc finger proteins specific for the polypeptide: NH2-Met-Asp-Ile-Ser-Ser-Ala-Ser- His-Phe-Pro-Leu-Glu-Val-Glu-Ala-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。 The polypeptide respectively formed limpet hemocyanin and bovine serum albumin coupled complex blood, see methods: 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。 4mg hemocyanin using the above-described polypeptide complex together with complete Freund's adjuvant to immunize rabbits, 15 days and then hemocyanin polypeptide complex plus incomplete Freund's adjuvant boosted once. 采用经15μg/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。 Employed by 15μg / ml bovine serum albumin polypeptide complex coated ELISA assay plates made of rabbit serum antibody titers. 用蛋白A-Sepharose从抗体阳性的家兔血清中分离总IgG。 Total IgG isolated from the serum of rabbit antibodies with Protein A-Sepharose. 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。 The polypeptide bound to cyanogen bromide activated Sepharose4B column, by affinity chromatography from total IgG isolated anti-polypeptide antibody. 免疫沉淀法证明纯化的抗体可特异性地与人锌指蛋白8.8结合。 Immunoprecipitation of purified antibodies specifically demonstrated with human zinc finger protein binding 8.8. 实施例6:本发明的多核苷酸片段用作杂交探针的应用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。 Example 6: a polynucleotide fragment of the invention as the application of the hybridization probe selected from the polynucleotides of the present invention of a suitable oligonucleotide fragments used as hybridization probes has many uses, such as the probe needle may be genomic or cDNA library hybridized to normal tissues or pathological tissues from different sources in order to identify whether it contains a polynucleotide sequence and polynucleotide sequences homologous to the detection of the present invention, the probe can also be used for further testing of the present invention. polynucleotide sequence or a polynucleotide sequence homologous to the expression in normal tissues or pathological tissues and 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。 Purpose of this example is from a polynucleotide of the present invention SEQ ID NO: 1 selected if it contains a polynucleotide according to the present invention, some tissues suitable oligonucleotide fragments used as hybridization probes, filter hybridization and identified by methods acid sequence or a homologous polynucleotide sequences. 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。 Filter hybridization methods include dot blotting, Southern blotting, Northern blotting and other copying methods, polynucleotide sample to be tested which are fixed to substantially the same procedure used in the hybridization on the filter. 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。 These steps are the same: a fixed first sample filter prehybridization using hybridization buffer without probe, so that the non-specific binding sites on the filter carrier and the sample is saturated with synthetic polymers. 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。 Then the prehybridization solution containing probe labeled hybridization buffer is replaced, and incubated with the probe hybridize to the target nucleic acid. 杂交步骤之后,未杂交上的探针被一系列洗膜步骤除掉。 After hybridization step, the probe is not hybridized membrane was washed a series of steps to get rid of. 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。 This embodiment utilizes the membrane was washed higher strength conditions (e.g., lower salt concentration and higher temperature), and so reduce background hybridization specificity retain only strong signals. 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。 Selection of the embodiment of the probe embodiment comprises two categories: the polynucleotide probe is completely present invention SEQ ID NO: identical or complementary to oligonucleotides 1; the second probe is a part of the present invention polynucleotide of SEQ ID NO: identical or complementary to an oligonucleotide fragment. 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。 Selection Example of the present embodiment Dot Blot Samples were fixed to the membrane, the membrane was washed under conditions of high intensity, a first type-specific hybridization probe and the sample is retained with the strongest. 一、探针的选用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:1,探针大小优选范围为18-50个核苷酸;2,GC含量为30%-70%,超过则非特异性杂交增加; A probe selected from the polynucleotides of the present invention SEQ ID NO: 1, selection of an oligonucleotide fragments used as hybridization probes, the following principles should be followed and several aspects need to be considered: 1, preferably size range of the probe 18-50 nucleotides; 2, GC content of 30% -70%, than the increase in non-specific hybridization;

3,探针内部应无互补区域;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;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。 3, the probe should be no internal complementary region; 4, can meet the above criteria as a primary probe, and a computer for further sequence analysis, the primary probe comprises a sequence region which they are derived, respectively (i.e., SEQ ID NO: 1) and other known genomic sequence and the complementary region of homology comparison, if non-target molecules with homology region is greater than 85% or more than 15 contiguous bases identical to, the primary probe is generally not it should be used; 5, the final selection of the primaries whether the probe as the probe has practical value should be further determined by experiment.

完成以上各方面的分析后挑选并合成以下二个探针:探针1(probe1),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):5'-TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG-3'(SEQ ID NO:8)探针2(probe2),属于第二类探针,相当于SEQ ID NO:1的基因片段或其互补片段的替换突变序列(41Nt):5'-TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG-3'(SEQ ID NO:9)与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文献:DNA PROBES GHKeller;MMManak;Stockton Press,1989(USA)以及更常用的分子克隆实验手册书籍如《分子克隆实验指南》(1998年第二版)[美]萨姆布鲁克等著,科学出版社。 After completion of the above aspects of the analysis and selection of the following two synthetic probes: Probe 1 (PROBE1), belonging to the first probe, and SEQ ID NO: 1 gene fragment is fully complementary or homologous (41Nt): 5 ' -TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG-3 '(SEQ ID NO: 8) probe 2 (probe2), belong to the second probe, corresponding to SEQ ID NO: 1, or a replacement gene fragment complementary to a fragment of the mutant sequence (41Nt): 5' -TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG-3 '(SEQ ID NO: 9) and other conventional formulating agents not listed a method associated with the following specific experimental procedure refer to Document: DNA PROBES GHKeller; MMManak; Stockton Press, 1989 (USA), and more commonly molecular cloning a Laboratory Manual books such as "molecular cloning a Laboratory Manual" (second edition 1998) [US] Sambrook waiting, Science Press.

样品制备:1,从新鲜或冰冻组织中提取DNA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。 Sample preparation: 1, the step of extracting DNA from fresh or frozen tissues: a) the fresh or freshly thawed normal liver tissue dip into ice and filled with phosphate-buffered saline (PBS) in a petri dish. 用剪刀或手术刀将组织切成小块。 With scissors or a scalpel tissue cut into small pieces. 操作中应保持组织湿润。 Operation tissue should be kept moist. 2)以1000g离心切碎组织10分钟。 2) minced tissue was centrifuged at 1000g for 10 minutes. 3)用冷匀浆缓冲液(0.25mol/L蔗糖;25mmol/LTris-HCl,pH7.5;25mmol/LnaCl;25mmol/L MgCl2)悬浮沉淀(大约10ml/g)。 3) cold homogenization buffer (0.25mol / L sucrose; 25mmol / LTris-HCl, pH7.5; 25mmol / LnaCl; 25mmol / L MgCl2) was suspended precipitate (approximately 10ml / g). 4)在4℃用电动匀浆器以全速匀浆组织悬液,直至组织被完全破碎。 4) it is completely broken at 4 deg.] C with an electric tissue homogenizer suspension homogenized at full speed, until the tissue. 5)1000g离心10分钟。 5) 1000g centrifugation for 10 min. 6)用重悬细胞沉淀(每0.1g最初组织样品加1-5ml),再以1000g离心10分钟。 6) Resuspend the cell pellet (original tissue per 0.1g sample adding 1-5ml), and then centrifuged at 1000g for 10 min. 7)用裂解缓冲液重悬沉淀(每0.1g最初组织样品加1ml),然后接以下的苯酚抽提法。 7) with lysis buffer and resuspend pellet (0.1g per tissue sample was originally added 1ml), then take the following phenol extraction method. 2,DNA的苯酚抽提法步骤:1)用1-10ml冷PBS洗细胞,1000g离心10分钟。 2, DNA was phenol extraction method steps: 1) the cells were washed with 1-10ml of cold PBS, centrifuged at 1000g for 10 minutes. 2)用冷细胞裂解液重悬浮沉淀的细胞(1×108细胞/ml)最少应用100ul裂解缓冲液。 2) with cold cell lysate pelleted cells were resuspended (1 × 108 cells / ml) Application least 100ul lysis buffer. 3)加SDS至终浓度为1%,如果在重悬细胞之前将SDS直接加入到细胞沉淀中,细胞可能会形成大的团块而难以破碎,并降低的总产率。 3) SDS was added to a final concentration of 1%, if the cells were resuspended before SDS was added directly to the cell pellet, the cells may be difficult to form large agglomerates crushed, and reduced overall yield. 这一点在抽提>107细胞时特别严重。 In this stripping> 107 cells particularly severe. 4)加蛋白酶K至终浓度200ug/ml。 4) Protease K was added to a final concentration of 200ug / ml. 5)50℃保温反应1小时或在37℃轻轻振摇过夜。 5) 50 ℃ incubated gently shaking for 1 hour or overnight at 37 ℃. 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。 6) with an equal volume of phenol: chloroform: isoamyl alcohol (25:24) extraction, centrifugation in microcentrifuge tube for 10 minutes. 两相应清楚分离,否则重新进行离心。 Clear separation of the corresponding two, or re-centrifuged. 7)将水相转移至新管。 7) The aqueous phase was transferred to a new tube. 8)用等体积氯仿∶异戊醇(24∶1)抽提,离心10分钟。 8) with an equal volume of chloroform: isoamyl alcohol (24:1) extracted, centrifuged for 10 minutes. 9)将含DNA的水相转移至新管。 9) The DNA-containing aqueous phase transferred to a new tube. 然后进行DNA的纯化和乙醇沉淀。 Followed by DNA purification and ethanol precipitation. 3,DNA的纯化和乙醇沉淀步骤: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。 . 3, DNA purification and ethanol precipitation steps: 1) A 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol was added to the DNA solution and mix. 在-20℃放置1小时或至过夜。 To stand for 1 hour or overnight at -20 ℃. 2)离心10分钟。 2) for 10 minutes. 3)小心吸出或倒出乙醇。 3) Carefully aspirated or decanted ethanol. 4)用70%冷乙醇500ul洗涤沉淀,离心5分钟。 4) wash with 70% cold ethanol precipitation 500ul, centrifuged for 5 minutes. 5)小心吸出或倒出乙醇。 5) carefully aspirated or decanted ethanol. 用500ul冷乙醇洗涤沉淀,离心5分钟。 500ul precipitate was washed with cold ethanol, centrifuged for 5 minutes. 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。 6) carefully aspirated or decanted ethanol, ethanol and the residue was then inverted onto absorbent paper shed. 空气干燥10-15分钟,以使表面乙醇挥发。 Air dried for 10-15 minutes, so that the surface to evaporate the ethanol. 注意不要使沉淀完全干燥,否则较难重新溶解。 Be careful not to precipitate completely dry, otherwise it is difficult to re-dissolve. 7)以小体积TE或水重悬DNA沉淀。 7) in a small volume of water or TE resuspended DNA pellet. 低速涡旋振荡或用滴管吹吸,同时逐渐增加TE,混合至DNA充分溶解,每1-5×106细胞所提取的大约加1ul。 Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, × 106 cells per plus about 1-5 extracted 1ul.

以下第8-13步骤仅用于必须除去污染时,否则可直接进行第14步骤。 The following steps 8-13 only when contaminated, can be directly or step 14 must be removed. 8)将RNA酶A加到DNA溶液中,终浓度为100ug/ml,37℃保温30分钟。 8) The RNA enzyme A was added to the DNA solution to a final concentration of 100ug / ml, incubated 37 ℃ 30 minutes. 9)加入SDS和蛋白酶K,终浓度分别为0.5%和100ug/ml。 9) addition of SDS and proteinase K, a final concentration of 0.5% and 100ug / ml. 37℃保温30分钟。 37 [deg.] C for 30 minutes. 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。 10) with an equal volume of phenol: chloroform: isoamyl alcohol (25:24) The reaction solution was extracted, centrifuged for 10 minutes. 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。 11) The aqueous phase was carefully removed with an equal volume of chloroform: isoamyl alcohol (24:1) re-extracted, centrifuged for 10 minutes. 12)小心移出水相,加1/10体积2mol/L醋酸钠和2.5体积冷乙醇,混匀置-20℃ 1小时。 12) The aqueous phase was carefully removed, added 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, mix set to -20 ℃ 1 hour. 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。 13) was washed with 70% ethanol and precipitated with 100% ethanol, air dried, resuspended nucleic acid, the same procedure of step 3-6. 14)测定A260和A280以检测DNA的纯度及产率。 14) Determination of A260 and A280 in purity and yield of DNA detection. 15)分装后存放于-20℃。 15) aliquots stored at -20 ℃. 样膜的制备:1)取4×2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。 The film samples were prepared: 1) Take a nitrocellulose membrane 4 × 2 Zhang appropriate size (NC membrane), marked with a pencil point-like location and gently like numbers thereon, each probe required two NC membrane, washing the membrane so as to respectively condition and high strength of the strength conditions after the experimental procedure. 2)吸取及对照各15微升,点于样膜上,在室温中晾干。 2) 15 microliters of each draw and control, point to the sample film, dried at room temperature. 3)置于浸润有0.1mol/LNaOH,1.5mol/LNaCl的滤纸上5分钟(两次),晾干置于浸润有0.5mol/L Tris-HCl(pH7.0),3mol/LNaCl的滤纸上5分钟(两次),晾干。 3) is placed impregnated with 0.1mol / LNaOH, 5 minutes (twice on 1.5mol / LNaCl filter paper), impregnated with dried was placed on 0.5mol L Tris-HCl (pH7.0), 3mol / LNaCl filter paper / 5 minutes (twice), dried. 4)夹于干净滤纸中,以铝箔包好,60-80℃真空干燥2小时。 4) interposed in a clean filter paper, wrapped in aluminum foil, and dried under vacuum 60-80 ℃ 2 hours.

探针的标记1)3μlProbe(0.10D/10μl),加入2μlKinase缓冲液,8-10uCiγ-32P-dATP+2UKinase,以补加至终体积20μl。 The labeled probe 1) 3μlProbe (0.10D / 10μl), was added 2μlKinase buffer, 8-10uCiγ-32P-dATP + 2UKinase, supplemented to a final volume of 20μl. 2)37℃保温2小时。 2) 37 ℃ for 2 hours. 3)加1/5体积的溴酚蓝指示剂(BPB)。 3) Add 1/5 volume indicator bromophenol blue (BPB). 4)过Sephadex G-50柱。 4) through 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) to have a first 32P-Probe begin collecting peak (monitoring Monitor available) before washing out. 6)5滴/管,收集10-15管。 6) 5 drops / tube, the collection tube 10-15. 7)用液体闪烁仪监测同位素量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)。 7) was monitored using a liquid scintillation isotopes 8) were collected after the first peak were combined to prepare the desired 32P-Probe (second peak as the free γ-32P-dATP).

预杂交将样膜置于塑料袋中,加入3-10mg预杂交液(10×Denhardt's;6×SSC,0.1mg/mlCT DNA(小牛胸腺DNA)。),封好袋口后,68℃水浴摇2小时。 Prehybridization The sample film was placed in a plastic bag, is added 3-10mg pre-hybridization solution (10 × Denhardt's;. 6 × SSC, 0.1mg / mlCT DNA (the DNA calf thymus)), the sealed bag, a water bath at 68 deg.] C shake for two 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℃水浴摇过夜。 The plastic bag corner cut hybridization, probes prepared was added, the sealed bag, 42 ℃ shaking water bath overnight.

洗膜:高强度洗膜:1)取出已杂交好的样膜。 The membrane was washed: high-strength membrane was washed: 1) Remove the film samples have good hybridization.

2)2×SSC,0.1%SDS中,40℃洗15分钟(2次)。 2) 2 × SSC, 0.1% SDS in, 40 ℃ wash for 15 minutes (2 times).

3)0.1×SSC,0.1%SDS中,40℃洗15分钟(2次)。 3) 0.1 × SSC, 0.1% SDS in, 40 ℃ wash for 15 minutes (2 times).

4)0.1×SSC,0.1%SDS中,55℃洗30分钟(2次),室温晾干。 4) 0.1 × SSC, 0.1% SDS, the wash for 30 minutes (2 times) 55 deg.] C, dried at room temperature. 低强度洗膜:1)取出已杂交好的样膜。 Washing the membrane strength is low: 1) Remove the film samples have good hybridization.

2)2×SSC,0.1%SDS中,37℃洗15分钟(2次)。 2) 2 × SSC, 0.1% SDS in, 37 ℃ wash for 15 minutes (2 times).

3)0.1×SSC,0.1%SDS中,37℃洗15分钟(2次)。 3) 0.1 × SSC, 0.1% SDS in, 37 ℃ wash for 15 minutes (2 times).

4)0.1×SSC,0.1%SDS中,40℃洗15分钟(2次),室温晾干。 4) 0.1 × SSC, 0.1% SDS in, 40 deg.] C wash for 15 minutes (2 times), dried at room temperature.

X-光自显影:-70℃,X-光自显影(压片时间根据杂交斑放射性强弱而定)。 X- ray autoradiography: -70 ℃, X- ray autoradiography (tableting spot time according to radioactive hybridization intensity).

实验结果: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;而采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。 Experimental Results: Experiments using a low intensity hybridization wash conditions for the film, two or more probes hybridize to the radioactive spots no significant difference in strength; and the use of high strength hybridization experiments carried out to wash the film forming conditions, plaque hybridization probes radioactivity 1 radioactivity was significantly stronger than other plaques hybridized to the probe. 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。 Thus the probe can be used to qualitatively and quantitatively analyze a polynucleotide of the present invention, the presence and differential expression in different tissues. 实施例7 DNA Microarray基因芯片或基因微矩阵(DNA Microarray)是目前许多国家实验室和大制药公司都在着手研制和开发的新技术,它是指将大量的靶基因片段有序地、高密度地排列在玻璃、硅等载体上,然后用荧光检测和计算机软件进行数据的比较和分析,以达到快速、高效、高通量地分析生物信息的目的。 Example 7 DNA Microarray gene chip or microarray gene (DNA Microarray) is currently implemented in many national laboratories and pharmaceutical companies are working on new technology research and development, which refers to the large number of target gene fragment in an orderly manner, high density arranged on a glass, silicon or the like support, and then analyzed and compared with the fluorescence detection data and computer software, in order to achieve fast, efficient, high-throughput analysis of biological information purposes. 本发明的多核苷酸可作为靶DNA用于基因芯片技术用于高通量研究新基因功能;寻找和筛选组织特异性新基因特别是肿瘤等疾病相关新基因;疾病的诊断,如遗传性疾病。 Polynucleotides of the present invention can be used as target DNA gene chip technology for high throughput functional study of new genes; specific gene and screening to find a particular tissue diseases such as cancer related gene; diagnosis of diseases, such as hereditary diseases . 其具体方法步骤在文献中已有多种报道,如可参阅文献DeRisi,JL,Lyer,V.&Brown,PO(1997)Science278,680-686.及文献Helle,RA,Schema,M.,Chai,A.,Shalom,D.,(1997)PNAS 94:2150-2155.(一)点样各种不同的全长cDNA共计4000条多核苷酸序列作为靶DNA,其中包括本发明的多核苷酸。 The specific method steps have been reported in the literature more, as may be found in the literature DeRisi, JL, Lyer, V & amp;.. Brown, PO (1997) Science278,680-686 and literature Helle, RA, Schema, M,. chai, A., Shalom, D, (1997) PNAS 94:.. 2150-2155 (a) spotting the nucleotide sequence of a total of more than 4000 different full-length cDNA as target DNA, which comprises a polynucleotide of the present invention acid. 将它们分别通过PCR进行扩增,纯化所得扩增产物后将其浓度调到500ng/ul左右,用Cartesian 7500点样仪(购自美国Cartesian公司)点于玻璃介质上,点与点之间的距离为280μm。 They were amplified by the PCR, which amplification product obtained was purified after concentration was adjusted to about 500ng / ul, with Cartesian spotter 7500 (available from Cartesian Corporation USA) the point on the glass medium between the dots distance of 280μm. 将点样后的玻片进行水合、干燥、置于紫外交联仪中交联,洗脱后干燥使DNA固定在玻璃片上制备成芯片。 After spotting the slides hydrated, dried, UV cross-linking apparatus is placed in a crosslinked, dried DNA was prepared into chips fixed on the glass sheet after elution. 其具体方法步骤在文献中已有多种报道,本实施例的点样后处理步骤是:1.潮湿环境中水合4小时;2.0.2%SDS洗涤1分钟;3.ddH2O洗涤两次,每次1分钟;4.NaBH4封闭5分钟;5.95℃水中2分钟;6.0.2%SDS洗涤1分钟;7.ddH2O冲洗两次;8.凉干,25℃储存于暗处备用。 The specific method steps have been various reports in the literature, after spotting procedure of this embodiment of the process is: 1 4 hours wet in the hydrated environment; washed with 2.0.2% SDS 1 minutes; 3.ddH2O washed twice, each times 1 minute; 4.NaBH4 blocked for 5 minutes; 2 minutes 5.95 ℃ water; washed with 6.0.2% SDS 1 min; 7.ddH2O rinsed twice;. 8 to dry, 25 ℃ backup stored in the dark. (二)探针标记用一步法分别从人体混合组织与机体特定组织(或经过刺激的细胞株)中抽提总mRNA,并用Oligotex mRNA Midi Kit(购自QiaGen公司)纯化mRNA,通过反转录分别将荧光试剂Cy3dUTP(5-Amino-propargyl-2'-deoxyuridine 5'-triphatecoupled to Cy3 fluorescent dye,购自Amersham Phamacia Biotech公司)标记人体混合组织的mRNA,用荧光试剂Cy5dUTP(5-Amino-propargyl-2'-deoxyuridine5'-triphate coupled to Cy5 fluorescent dye,购自Amer sham Phamacia Biotech公司)标记机体特定组织(或经过刺激的细胞株)mRNA,经纯化后制备出探针。 (B) labeled probes were extracted from the body with a one-step mixing with body tissue specific tissue (or cell line after stimulation) total mRNA, and (available from QiaGen Inc.) mRNA was purified with Oligotex mRNA Midi Kit, by reverse transcription the fluorescent reagent were Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphatecoupled to Cy3 fluorescent dye, commercially available from Amersham Phamacia Biotech company) mixing human tissue mRNA labeled with a fluorescent reagent Cy5dUTP (5-Amino-propargyl- 2'-deoxyuridine5'-triphate coupled to Cy5 fluorescent dye, available from Amer sham Phamacia Biotech company) labeled specific body tissue (or cell line after stimulation) mRNA, a probe was prepared after purification. 具体步骤参照及方法见:Schena,M.,Shalon,D.,Heller,R.(1996)Proc.Natl.Acad.Sci.USA.Vol.93:10614-10619.Schena,M.,Sbalon,Dari.,Dayis,RW(1995)ScienGe.2 70.(20):467-480.(三)杂交分别将来自以上两种组织的探针与芯片一起在UniHybTMHybridizationSolution(购自TeleChem公司)杂交液中进行杂交16小时,室温用洗涤液(1×SSC,0.2%SDS)洗涤后用ScanArray 3000扫描仪(购自美国General Scanning公司)进行扫描,扫描的图象用Imagene软件(美国Biodiscovery公司)进行数据分析处理,算出每个点的Cy3/Cy5比值。 Referring to the specific steps and methods, see: Schena, M., Shalon, D., Heller, R (1996) Proc.Natl.Acad.Sci.USA.Vol.93:. 10614-10619.Schena, M., Sbalon, Dari ., Dayis, RW (1995) ScienGe.2 70. (20):. 467-480 (three) were hybridized with a probe from the chip together two or more tissue UniHybTMHybridizationSolution (available from TeleChem Inc.) hybridization solution hybridization for 16 hours at room temperature with a washing solution (1 × SSC, 0.2% SDS) was washed with (commercially available from General scanning Corporation US) scanned with ScanArray 3000 scanner, image data using Imagene analysis software (Biodiscovery Corporation USA) processing, calculating each point Cy3 / Cy5 ratios.

以上机体特定组织(或经过刺激的细胞株)分别为胎脑、膀胱粘膜、PMA+的Ecv304细胞株、LPS+的Ecv304细胞株胸腺、正常成纤维细胞1024NC、Fibroblast,生长因子刺激,1024NT、疤痕成fc生长因子刺激,1013HT、疤痕成fc未用生长因子刺激,1013HC、膀胱癌建株细胞EJ、膀胱癌旁、膀胱癌、肝癌、肝癌细胞株、胎皮、脾脏、前列腺癌、空肠腺癌、贲门癌。 The above body a particular tissue (or via stimulated cell line) were fetal brain, bladder mucosa, PMA + of Ecv304 cell lines, LPS + of Ecv304 cell lines thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scars into fc growth factor stimulation, 1013HT, scar with growth factors to stimulate not fc, 1013HC, EJ bladder carcinoma cell line construction, adjacent bladder cancer, bladder cancer, hepatoma, hepatocellular carcinoma cell lines, fetal skin, spleen, prostate cancer, adenocarcinoma of the jejunum, cardia cancer. 根据这18个Cy3/Cy5比值绘出折方图。 18 according to this Cy3 / Cy5 ratios drawn off side in FIG. (图1)。 (figure 1). 由图可见本发明所述的人锌指蛋白8.8和人锌指蛋白表达谱很相似。 The present invention is seen in FIG human zinc finger protein 8.8 and human zinc finger protein expression profiles very similar.

序列表(1)一般信息:(ii)发明名称:人锌指蛋白8.8及其编码序列(iii)序列数目:9(2)SEQ ID NO:1的信息:(i)序列特征:(A)长度:716bp(B)类型:核酸(C)链性:双链(D)拓扑结构:线性(ii)分子类型:cDNA(xi)序列描述:SEQ ID NO:1:1 GAAGCACTTTCTTAAGACTGACCTAACGCTGGATTATTTCCCGTCCAATGCCTGCATGCT61 GCTTGAATTGCTCCACCCACACCTCCATGACCAAGGGCGCCAGAGTGCTGCAACTGGGGC121 GTGGGCCGCTCTCTGCTTTTCCTGTCTGACTCTGACAAGTCCTCCCTCACTGAATGTAGA181 ATCGTTGCCAAGTTTCTGAGAAGTGTCGATTCCCTGTTAGCATGGATATCAGTTCTGCCT241 CACATTTCCCACTTGAGGTTGAGGCGTACTGGAGACAACACCTCAGACCATCTGAACCCC301 ATCAGTGGATGAAAATGGGGCTGTTAATATACTCTAAAAGCCATACTAAAAATGCTCTGA361 GGGAACTGGCTAAGAATAGTGGGCCTGGTGATTGTCTATCACGCAAGGCTTTGTTTTGTA421 CTGTTCAGAAATCTGTCACCTTTCTGCCTGCCCTTGTTTCCTGAATGAAATGCTTCTGGG481 GTTATTTATGAAAGGAGTGATCCTGGGGCAGGCAGGAGGCAGTGGGCTTCATGGCTCCTT541 GAAGTTATTACTGATCTTGACCTTCTCTTTGGCTACCTTTAGACAAAGAATACGCCAATC601 AATACTTGGGGCTCTAAGTTTTACAATTGATATTTATTTGTATCATCTCTTTGTCTAGGA661 SEQUENCE LISTING (1) General information: (II) Title: Human zinc finger protein 8.8 the number of sequences and coding sequences (iii): 9 (2) SEQ ID NO: 1 Information: (i) SEQUENCE CHARACTERISTICS: (A) length: 716bp (B) tYPE: nucleic acid (C) strandedness: double stranded (D) tOPOLOGY: linear (ii) mOLECULE tYPE: cDNA (xi) sEQUENCE dESCRIPTION: SEQ ID NO: 1: 1 GAAGCACTTTCTTAAGACTGACCTAACGCTGGATTATTTCCCGTCCAATGCCTGCATGCT61 GCTTGAATTGCTCCACCCACACCTCCATGACCAAGGGCGCCAGAGTGCTGCAACTGGGGC121 GTGGGCCGCTCTCTGCTTTTCCTGTCTGACTCTGACAAGTCCTCCCTCACTGAATGTAGA181 ATCGTTGCCAAGTTTCTGAGAAGTGTCGATTCCCTGTTAGCATGGATATCAGTTCTGCCT241 CACATTTCCCACTTGAGGTTGAGGCGTACTGGAGACAACACCTCAGACCATCTGAACCCC301 ATCAGTGGATGAAAATGGGGCTGTTAATATACTCTAAAAGCCATACTAAAAATGCTCTGA361 GGGAACTGGCTAAGAATAGTGGGCCTGGTGATTGTCTATCACGCAAGGCTTTGTTTTGTA421 CTGTTCAGAAATCTGTCACCTTTCTGCCTGCCCTTGTTTCCTGAATGAAATGCTTCTGGG481 GTTATTTATGAAAGGAGTGATCCTGGGGCAGGCAGGAGGCAGTGGGCTTCATGGCTCCTT541 GAAGTTATTACTGATCTTGACCTTCTCTTTGGCTACCTTTAGACAAAGAATACGCCAATC601 AATACTTGGGGCTCTAAGTTTTACAATTGATATTTATTTGTATCATCTCTTTGTCTAGGA661 ATGTAAAAGTGATTCTAAACTAAGATGTGTAATAAAAATCAATCAGATTTATTGTA(3)SEQ ID NO:2的信息:(i)序列特征:(A)长度:80个氨基酸(B)类型:氨基酸(D)拓扑结构:线性(ii)分子类型:多肽(xi)序列描述:SEQ ID NO:2:1 Met Asp Ile Ser Ser Ala Ser His Phe Pro Leu Glu Val Glu Ala16 Tyr Trp Arg Gln His Leu Arg Pro Ser Glu Pro His Gln Trp Met31 Lys Met Gly Leu Leu Ile Tyr Ser Lys Ser His Thr Lys Asn Ala46 Leu Arg Glu Leu Ala Lys Asn Ser Gly Pro Gly Asp Cys Leu Ser61 Arg Lys Ala Leu Phe Cys Thr Val Gln Lys Ser Val Thr Phe Leu76 Pro Ala Leu Val Ser(4)SEQ ID NO:3的信息(i)序列特征(A)长度:24碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii)分子类型:寡核苷酸(xi)序列描述:SEQ ID NO:3:GAAGCACTTTCTTAAGACTGACCT 24(5)SEQ ID NO:4的信息(i)序列特征(A)长度:24碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii) ATGTAAAAGTGATTCTAAACTAAGATGTGTAATAAAAATCAATCAGATTTATTGTA (3) SEQ ID NO: 2 information: (i) SEQUENCE CHARACTERISTICS: (A) length: 80 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) Sequence description: SEQ ID NO: 2: 1 Met Asp Ile Ser Ser Ala Ser His Phe Pro Leu Glu Val Glu Ala16 Tyr Trp Arg Gln His Leu Arg Pro Ser Glu Pro His Gln Trp Met31 Lys Met Gly Leu Leu Ile Tyr Ser Lys Ser His Thr Lys Asn Ala46 Leu Arg Glu Leu Ala Lys Asn Ser Gly Pro Gly Asp Cys Leu Ser61 Arg Lys Ala Leu Phe Cys Thr Val Gln Lys Ser Val Thr Phe Leu76 Pro Ala Leu Val Ser (4) SEQ ID NO: 3 information ( i) sEQUENCE cHARACTERISTICS (A) length: 24 bases (B) tYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) mOLECULE tYPE: oligonucleotide (xi) sequence dESCRIPTION: SEQ ID NO: 3: GAAGCACTTTCTTAAGACTGACCT 24 (5) SEQ ID NO: 4 sEQUENCE cHARACTERISTICS information (i) (a) length: 24 bases (B) tYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) 分子类型:寡核苷酸(xi)序列描述:SEQ ID NO:4:TACAATAAATCTGATTGATTTTTA 24(6)SEQ ID NO:5的信息(i)序列特征(A)长度:33碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii)分子类型:寡核苷酸(xi)序列描述:SEQ ID NO:5:CCCCATATGATGGATATCAGTTCTGCCTCACAT 33(7)SEQ ID NO:6的信息(i)序列特征(A)长度:33碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii)分子类型:寡核苷酸(xi)序列描述:SEQ ID NO:6:CATGGATCCTCAGGAAACAAGGGCAGGCAGAAA 33(8)SEQ ID NO:7的信息:(i)序列特征:(A)长度:15个氨基酸(B)类型:氨基酸(D)拓扑结构:线性(ii)分子类型:多肽(xi)序列描述:SEQ ID NO:7:Met-Asp-Ile-Ser-Ser-Ala-Ser-His-Phe-Pro-Leu-Glu-Val-Glu-Ala 15(9)SEQ ID NO:8的信息(i)序列特征(A)长度:41碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii)分子类型:寡核苷酸(xi)序列 MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TACAATAAATCTGATTGATTTTTA 24 (6) SEQ ID NO: wherein the sequence information (i) 5 (A), length: 33 bases (B) TYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) mOLECULE tYPE: oligonucleotide (xi) sEQUENCE dESCRIPTION: SEQ ID NO: 5: CCCCATATGATGGATATCAGTTCTGCCTCACAT 33 (7) SEQ ID NO: 6 information ( i) sEQUENCE cHARACTERISTICS (A) length: 33 bases (B) tYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) mOLECULE tYPE: oligonucleotide (xi) sequence dESCRIPTION: SEQ ID NO: 6: CATGGATCCTCAGGAAACAAGGGCAGGCAGAAA 33 (8) SEQ ID NO: information 7: (i) sEQUENCE cHARACTERISTICS: (a) length: 15 amino acids (B) tYPE: amino acid (D) tOPOLOGY: linear (ii) mOLECULE tYPE : polypeptide (xi) sEQUENCE dESCRIPTION: SEQ ID NO: 7: Met-Asp-Ile-Ser-Ser-Ala-Ser-His-Phe-Pro-Leu-Glu-Val-Glu-Ala 15 (9) SEQ ID NO : characteristic information sequence (i) 8 (a), length: 41 bases (B) tYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) mOLECULE tYPE: oligonucleotide (xi )sequence 述:SEQ ID NO:8:TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG 41(10)SEQ ID NO:9的信息(i)序列特征(A)长度:41碱基(B)类型:核酸(C)链性:单链(D)拓扑结构:线性(ii)分子类型:寡核苷酸(xi)序列描述:SEQ ID NO:9:TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG 41 Said: SEQ ID NO: 8: TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG 41 (10) SEQ ID NO: wherein the sequence information (i) 9 (A), length: 41 bases (B) TYPE: nucleic acid (C) strandedness: single strand (D) tOPOLOGY: linear (ii) mOLECULE tYPE: oligonucleotide (xi) sEQUENCE dESCRIPTION: SEQ ID NO: 9: TGGATATCAGTTCTGCCTCACATTTCCCACTTGAGGTTGAG 41

Claims (18)

  1. 1.一种分离的多肽-人锌指蛋白8.8,其特征在于它包含有:SEQ ID NO:2所示的氨基酸序列的多肽、或其多肽的活性片段、类似物或衍生物。 1. An isolated polypeptide - human zinc finger protein 8.8, characterized in that it comprises: SEQ ID NO: amino acid sequence of the polypeptide shown in Figure 2, an active fragment or polypeptide, analogue or derivative thereof.
  2. 2.如权利要求1所述的多肽,其特征在于所述多肽、类似物或衍生物的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少95%的相同性。 2. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide, analog or derivative thereof having SEQ ID NO: 2 amino acid sequence at least 95% identity.
  3. 3.如权利要求2所述的多肽,其特征在于它包含具有SEQ ID NO:2所示的氨基酸序列的多肽。 Polypeptide according to claim 2, characterized in that it comprises a SEQ ID NO: amino acid sequence of the polypeptide shown in Figure 2.
  4. 4.一种分离的多核苷酸,其特征在于所述多核苷酸包含选自下组中的一种:(a)编码具有SEQ ID NO:2所示氨基酸序列的多肽或其片段、类似物、衍生物的多核苷酸;(b)与多核苷酸(a)互补的多核苷酸;或(c)与(a)或(b)有至少70%相同性的多核苷酸。 4. An isolated polynucleotide, wherein said polynucleotide comprises one selected from the group of: (a) encoding a SEQ ID NO: amino acid sequence of the polypeptide or fragment 2, like , a polynucleotide derivative; (b) a polynucleotide (a) a polynucleotide complementary to; or (c) to (a) or (b) at least 70% identical to a polynucleotide.
  5. 5.如权利要求4所述的多核苷酸,其特征在于所述多核苷酸包含编码具有SEQID NO:2所示氨基酸序列的多核苷酸。 As claimed in claim 4, wherein the polynucleotide, wherein the polynucleotide encoding having SEQID NO: 2 amino acid polynucleotide sequences.
  6. 6.如权利要求4所述的多核苷酸,其特征在于所述多核苷酸的序列包含有SEQ IDNO:1中80-464位的序列或SEQ ID NO:1中1-716位的序列。 1 bit sequence 80-464 or SEQ ID NO:: 1 in the sequence of positions 1-716 polynucleotide as claimed in claim 4, wherein said polynucleotide sequence comprising SEQ IDNO.
  7. 7.一种含有外源多核苷酸的重组载体,其特征在于它是由权利要求4-6中的任一权利要求所述多核苷酸与质粒、病毒或运载体表达载体构建而成的重组载体。 A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant vector constructed from the plasmid, virus or expressed from a carrier as claimed in claim any one of claims 4-6 said polynucleotide carrier.
  8. 8.一种含有外源多核苷酸的遗传工程化宿主细胞,其特征在于它是选自于下列一种宿主细胞:(a)用权利要求7所述的重组载体转化或转导的宿主细胞;或(b)用权利要求4-6中的任一权利要求所述多核苷酸转化或转导的宿主细胞。 A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is a host cell selected from one of the following: (a) a recombinant vector according to claim 7 with the host cell transformed or transduced ; or (b) any one of claims 4-6 with the polynucleotide as claimed in claim host cell transformed or transduced.
  9. 9.一种具有人锌指蛋白8.8活性的多肽的制备方法,其特征在于所述方法包括:(a)在表达人锌指蛋白8.8条件下,培养权利要求8所述的工程化宿主细胞;(b)从培养物中分离出具有人锌指蛋白8.8活性的多肽。 9. A method of preparing human zinc finger protein 8.8 polypeptide activity, characterized in that the method comprises: (a) expressing in the human zinc finger protein 8.8 conditions, culturing engineered host cells according to claim 8; (b) isolating from the culture was issued 8.8 zinc finger protein polypeptide activity.
  10. 10.一种能与多肽结合的抗体,其特征在于所述抗体是能与人锌指蛋白8.8特异性结合的抗体。 10. An antibody that binds to the polypeptide, wherein said antibody is capable of human zinc finger protein 8.8 antibody specifically binds.
  11. 11.一类模拟或调节多肽活性或表达的化合物,其特征在于它们是模拟、促进、拮抗或抑制人锌指蛋白8.8的活性的化合物。 11. A compound or a class of analog modulating polypeptide activity or expression, characterized in that they are analog, promote, antagonize or inhibit the activity of human zinc finger protein compound 8.8.
  12. 12.如权利要求11所述的化合物,其特征在于它是SEQ ID NO:1所示的多核苷酸序列或其片段的反义序列。 12. The compound according to claim 11, characterized in that it is SEQ ID NO: Antisense sequence of the polynucleotide sequences or fragments thereof shown in Table 1.
  13. 13.一种权利要求11所述化合物的应用,其特征在于所述化合物用于调节人锌指蛋白8.8在体内、体外活性的方法。 Application of the 11 13. A compound as claimed in claim, wherein the zinc finger compounds for regulating human body, the method of protein activity in vitro 8.8.
  14. 14.一种检测与权利要求1-3中的任一权利要求所述多肽相关的疾病或疾病易感性的方法,其特征在于其包括检测所述多肽的表达量,或者检测所述多肽的活性,或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。 1-3 in any one of claims related disease or susceptibility to a disease which is characterized in that the active polypeptide it comprises detecting the expression level of the polypeptide, or detecting the polypeptide of claim 14. A method of detecting and, or cause expression of the polypeptide amount or activity of nucleotide variations abnormality detecting polynucleotides.
  15. 15.如权利要求1-3中的任一权利要求所述多肽的应用,其特征在于它应用于筛选人锌指蛋白8.8的模拟物、激动剂,拮抗剂或抑制剂;或者用于肽指纹图谱鉴定。 15. The use of any one of claims 1-3, the polypeptide as claimed in claim claims, characterized in that it is applied to the screening of human zinc finger protein 8.8 mimetics, agonists, antagonists or inhibitors; or a peptide fingerprinting Pattern Method.
  16. 16.如权利要求4-6中的任一权利要求所述的核酸分子的应用,其特征在于它作为引物用于核酸扩增反应,或者作为探针用于杂交反应,或者用于制造基因芯片或微阵列。 16. The use of a nucleic acid molecule as claimed in claim any one of claims 4-6, characterized in that it as a primer for nucleic acid amplification reactions, or as probes for a hybridization reaction, or gene chip for producing or microarray.
  17. 17.如权利要求1-6及11中的任一权利要求所述的多肽、多核苷酸或化合物的应用,其特征在于用所述多肽、多核苷酸或其模拟物、激动剂、拮抗剂或抑制剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与人锌指蛋白8.8异常相关的疾病的药物组合物。 As claimed in any one of claims 1-6 and claim 11 according to claim polypeptide, polynucleotide or compound of the application, wherein with said polypeptide, polynucleotide or mimetics, agonists, antagonists inhibitor or a pharmaceutically safe and effective dose of a pharmaceutically acceptable carrier and as diagnostic or therapeutic pharmaceutical composition of human zinc finger diseases related to abnormal protein 8.8.
  18. 18.权利要求1-6及11中的任一权利要求所述的多肽、多核苷酸或化合物的应用,其特征在于用所述多肽、多核苷酸或化合物制备用于治疗如恶性肿瘤,血液病,HIV感染和免疫性疾病和各类炎症的药物。 1-6 and 18. The polypeptide of any one of claims 11 to claim, or the use of a compound of the polynucleotide, wherein the polypeptide with, polynucleotide or compound prepared for the treatment of malignant tumors, such as blood disease, HIV infection and drug immunological disease and various inflammation.
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