CN1198186U - Short form of chemokine beta-8 - Google Patents

Abstract

提供了人Ckβ－8截短形式的新型多肽。还提供了应用这些多肽的方法。A novel polypeptide in a truncated form of human Ckβ-8 is provided. Methods of using these polypeptides are also provided.

Description

趋化因子β-8的短形式Short form of chemokine beta-8

本发明涉及多肽和编码趋化因子β-8(Ckβ-8)的短形式的多核苷酸序列。具体地说，本发明涉及多核苷酸和多肽序列的应用，多核苷酸和多肽序列的生产，和抑制该多肽的作用。趋化因子也称为内泌性(intercrine)细胞因子，是结构上和功能上相关的细胞因子的亚族。这些分子都是小的诱导型促炎(proinflammatory)蛋白质。趋化因子在氨基酸水平一般表现为20％～75％的同源性，其特征是形成两个二硫键的4个保守半胱氨酸残基。基于前两个半胱氨酸残基的排列，趋化因子被分为两个亚族：α和β。在α亚族中，前两个半胱氨酸被一个氨基酸分开，于是表示为“C-X-C”亚族。在β亚族中，这两个半胱氨酸处于相邻位置，于是表示为“C-C”亚族。第一个半胱氨酸与第三个半胱氨酸形成一个二硫键，第二个与第四个也形成二硫键，导致许多趋化因子形成类似的三级结构。结构上，许多趋化因子经蛋白酶解加工而得功能性“成熟”蛋白质。这通常包括该蛋白质N端部分短前导序列的裂解。在蛋白质和/或cDNA水平已经描述了至少14种不同的α-趋化因子和12种β-趋化因子。The present invention relates to polypeptides and polynucleotide sequences encoding short forms of chemokine beta-8 (Ckbeta-8). In particular, the present invention relates to the use of polynucleotide and polypeptide sequences, the production of polynucleotide and polypeptide sequences, and the inhibition of the action of such polypeptides. Chemokines, also known as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are small inducible proinflammatory proteins. Chemokines generally exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines are divided into two subfamilies: alpha and beta. In the alpha subfamily, the first two cysteines are separated by an amino acid and are thus denoted the "C-X-C" subfamily. In the beta subfamily, these two cysteines are in adjacent positions, thus denoting the "C-C" subfamily. The first cysteine forms a disulfide bond with the third cysteine, and the second also forms a disulfide bond with the fourth, resulting in a similar tertiary structure for many chemokines. Structurally, many chemokines are proteolytically processed into functional "mature" proteins. This usually involves cleavage of the short leader sequence in the N-terminal portion of the protein. At least 14 different alpha-chemokines and 12 beta-chemokines have been described at the protein and/or cDNA level.

趋化因子表现出多种功能。一个重要特征是它们具有刺激不同种细胞如单核细胞、嗜中性白细胞、T淋巴细胞、嗜碱性粒细胞和成纤维细胞的趋化迁能的能力。α和β亚族在它们的细胞靶选择性方面有点不同。多数α-趋化因子吸引嗜中性白细胞、成纤维细胞、T细胞和NK细胞。β-趋化因子主要吸引单核细胞和T淋巴细胞。许多趋化因子具有促炎活性且在炎症反应期间包括于多个步骤。这些活性包括刺激组胺释放，溶酶体酶和白细胞三烯释放，增大靶免疫细胞与内皮细胞的粘附性，增强补体蛋白质的结合，诱导粒细胞粘着分子和补体受体的表达，和突发性呼吸。除了炎症中包括它们之外，一些趋化因子还表现出其它活性。例如，巨噬细胞炎性蛋白(MIP-1)能抑制造血干细胞增生，血小板因子-4(PF-4)可能是内皮细胞生长抑制剂，白细胞介素-8(IL-8)促进角质形成细胞增生，而GRO是黑素瘤细胞的自分泌生长因子。趋化因子与一些生理状态和疾病状况有关，特别是那些有炎性成分的病况。这些病况包括但不限于：淋巴细胞运输，创伤愈合，造血调节和免疫疾病如***反应、气喘和关节炎。许多趋化因子最初是作为炎症反应的产物分离的。例如，MIP-1最初是作为由巨噬细胞产生的内毒素诱导促炎细胞因子分离的。该亚族的其它成员已基于炎症诱导和氨基酸序列同源性而类似地进行了鉴定。这包括本发明的Ckβ-8多肽的全长形式和短形式。Chemokines exhibit multiple functions. An important feature is their ability to stimulate the chemotactic capacity of different types of cells such as monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. The alpha and beta subfamilies differ somewhat in their cellular target selectivity. Most alpha-chemokines attract neutrophils, fibroblasts, T cells and NK cells. Beta-chemokines mainly attract monocytes and T lymphocytes. Many chemokines have pro-inflammatory activity and are involved in multiple steps during the inflammatory response. These activities include stimulation of histamine release, release of lysosomal enzymes and leukotrienes, increased adhesion of target immune cells to endothelial cells, enhanced binding of complement proteins, induction of expression of granulocyte adhesion molecules and complement receptors, and Sudden breathing. In addition to their inclusion in inflammation, some chemokines exhibit other activities. For example, macrophage inflammatory protein (MIP-1) can inhibit hematopoietic stem cell proliferation, platelet factor-4 (PF-4) may be an inhibitor of endothelial cell growth, and interleukin-8 (IL-8) promotes keratinocytes proliferation, while GRO is an autocrine growth factor for melanoma cells. Chemokines are associated with a number of physiological states and disease states, especially those with an inflammatory component. These conditions include, but are not limited to, lymphocyte trafficking, wound healing, hematopoietic regulation, and immune diseases such as allergy, asthma, and arthritis. Many chemokines were originally isolated as products of an inflammatory response. For example, MIP-1 was originally isolated as an endotoxin-induced proinflammatory cytokine produced by macrophages. Other members of this subfamily have been similarly identified based on inflammation induction and amino acid sequence homology. This includes full-length and short forms of the Ckβ-8 polypeptides of the invention.

本发明一方面提供了人Ckβ-8的截短形式的新型多肽的混合物及其生物活性的和诊断上或治疗上有用的片段、类似物和衍生物。One aspect of the present invention provides mixtures of novel polypeptides of truncated forms of human Ckβ-8 and biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

本发明另一方面提供了编码这类多肽的分离核酸分子，包括mRNAs、DNAs、cDNAs、基因组DNA及其生物活性的和诊断上或治疗上有用的片段、类似物和衍生物。Another aspect of the invention provides isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA, and biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

本发明的又一方面提供了通过重组技术生产这类多肽的方法，这类重组技术包括：在促进表达所述蛋白质的条件下培养包括核酸序列的重组原核的和/或真核的宿主细胞，然后回收所述蛋白质。Yet another aspect of the present invention provides methods of producing such polypeptides by recombinant techniques comprising: culturing a recombinant prokaryotic and/or eukaryotic host cell comprising a nucleic acid sequence under conditions that promote expression of the protein, The protein is then recovered.

本发明的又一方面提供了将这类多肽或者编码这类多肽的多核苷酸用于治疗目的的方法，例如在化学疗法中保护骨髓干细胞以防化疗剂损伤，和刺激伤口愈合。Yet another aspect of the present invention provides methods of using such polypeptides or polynucleotides encoding such polypeptides for therapeutic purposes, such as in chemotherapy to protect bone marrow stem cells from damage by chemotherapeutic agents, and to stimulate wound healing.

本发明的又一方面提供了抗这种多肽的抗体。Yet another aspect of the present invention provides antibodies against such polypeptides.

本发明的又一方面提供了这种多肽的拮抗剂，它可用于抑制这种多肽的作用，例如，治疗再生障碍性贫血、脊髓发育不良综合征、气喘和关节炎。Yet another aspect of the present invention provides antagonists of such polypeptides, which are useful in inhibiting the effects of such polypeptides, eg, in the treatment of aplastic anemia, myelodysplastic syndrome, asthma and arthritis.

本发明的又一方面还提供了核酸探针，它包括与Ckβ-8核酸序列特异性地杂交的足够长的核酸分子。Yet another aspect of the present invention provides nucleic acid probes comprising nucleic acid molecules of sufficient length that specifically hybridize to the Ckβ-8 nucleic acid sequence.

本发明的又一方面提供了诊断分析法，用来检测与该多肽的表达不足和超量表达相关的疾病和检测编码这种多肽的核酸序列中的突变。Yet another aspect of the present invention provides diagnostic assays for detecting diseases associated with underexpression and overexpression of the polypeptide and for detecting mutations in nucleic acid sequences encoding such polypeptides.

本发明的又一方面提供了一种方法，该方法是为了开发治疗人类疾病的治疗剂和诊断剂，将这种多肽或者编码这种多肽的多核苷酸作为研究试剂用于与科研、DNA的合成和DNA载体的制备相关的体外目的。Yet another aspect of the present invention provides a method for developing therapeutic and diagnostic agents for treating human diseases, using the polypeptide or the polynucleotide encoding the polypeptide as a research reagent for use with scientific research, DNA In vitro purposes related to synthesis and preparation of DNA vectors.

通过本文的启示，本领域技术人员应能明白本发明的这些和其它方面。These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.

描述了编码在结构上与促炎“内泌性”趋化因子超家族相关的多肽的DNA序列。编码全长Ckβ-8(120个氨基酸)的多核苷酸序列得自主动脉的内皮cDNA文库。本发明涉及只包含长形式的82、76、75或74个C端氨基酸的Ckβ-8的新型截短形式。裂解了N端氨基酸和21个残基信号肽序列。就象Ckβ-8的长形式那样，这些克隆中的前两个半胱氨酸残基处于相邻位置，使它们属于趋化因子的“C-C”或β亚族。该基元的存在使它们有别于趋化因子的亚族(“CXC”)，“CXC”中前两个半胱氨酸残基被一个氨基酸分开。DNA sequences encoding polypeptides structurally related to the proinflammatory "endocrine" chemokine superfamily are described. The polynucleotide sequence encoding full-length Ck[beta]-8 (120 amino acids) was obtained from an endothelial cDNA library of the aorta. The present invention relates to novel truncated forms of Ckβ-8 comprising only the 82, 76, 75 or 74 C-terminal amino acids of the long form. The N-terminal amino acid and the 21-residue signal peptide sequence were cleaved. Like the long form of Ckβ-8, the first two cysteine residues in these clones are in adjacent positions, making them part of the "C-C" or β subfamily of chemokines. The presence of this motif distinguishes them from the subfamily of chemokines ("CXC") in which the first two cysteine residues are separated by one amino acid.

编码成熟多肽(SEQ ID NO.1、2、3和4)的多核苷酸序列可以用诸如RNA、ssDNA、基因组DNA等等的任何核酸形式来表示，它们包括与该多肽有关的其它编码序列和非编码序列。此外，由于氨基酸密码的简并性，编码相同的成熟多肽的任何DNA编码序列都应看作属于本发明的范围。还应包括这种序列等位的或非天然生成的任何变体。本发明还包括其中的编码序列在相同读框中与另一DNA序列融合的多核苷酸，该另一DNA序列帮助蛋白质从细胞表达或分泌或者作为标记物以鉴定细胞中的蛋白质(例如HA标记物)。A polynucleotide sequence encoding a mature polypeptide (SEQ ID NO. 1, 2, 3, and 4) can be represented by any nucleic acid form such as RNA, ssDNA, genomic DNA, etc., including other coding sequences associated with the polypeptide and noncoding sequences. Furthermore, due to the degeneracy of the amino acid code, any DNA coding sequence encoding the same mature polypeptide should be considered within the scope of the present invention. Also included are any allelic or non-naturally occurring variants of such sequences. The invention also includes polynucleotides in which the coding sequence is fused in the same reading frame to another DNA sequence that aids in the expression or secretion of a protein from a cell or serves as a marker to identify a protein in a cell (eg, an HA marker thing).

本发明还涉及与所述序列杂交的多核苷酸序列，它们至少具有50％、且优选为70％的序列相同性。这些多核苷酸编码保持与成熟多肽相同的生物功能或活性的多肽。该序列也可以是这类多核苷酸，即它们优选具有与本发明的序列相同并与之杂交的50个碱基，但未保留活性。这种序列可用作探针或PCR引物。The present invention also relates to polynucleotide sequences hybridizing to said sequences, which have at least 50%, and preferably 70% sequence identity. These polynucleotides encode polypeptides that retain the same biological function or activity as the mature polypeptide. The sequences may also be such polynucleotides, ie they preferably have 50 bases identical to and hybridize to the sequences of the present invention, but do not retain activity. Such sequences can be used as probes or PCR primers.

本发明的多肽可以是重组多肽、天然多肽或者合成多肽。这种多肽的衍生物指该多肽的下列情况：其中存在一个或多个氨基酸取代；其中一个或多个氨基酸包含取代基；其中成熟蛋白质与另一化合物融合；或者其中其它氨基酸与成熟蛋白质融合。The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides. A derivative of such a polypeptide refers to that polypeptide in which one or more amino acid substitutions are present; in which one or more amino acids contain a substituent; in which the mature protein is fused to another compound; or in which other amino acids are fused to the mature protein.

本发明的多肽优选呈分离形式提供，且优选纯化至均匀。The polypeptides of the invention are preferably provided in isolated form and are preferably purified to homogeneity.

本发明还涉及包括本发明的多核苷酸的载体，用该载体进行工程化的宿主细胞，和通过重组技术生产本发明的多肽。用本发明的载体对宿主细胞进行工程化(克隆或表达)。对本领域技术人员而言，为了激活启动子、选择转化体或者扩增截短了的Ckβ-8基因而采取的培养这些细胞的条件是显而易见的。The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells engineered with the vectors, and production of the polypeptides of the present invention by recombinant techniques. Host cells are engineered (cloned or expressed) with the vectors of the present invention. The conditions for culturing these cells for activating the promoter, selecting transformants, or amplifying the truncated Ckβ-8 gene will be apparent to those skilled in the art.

该多核苷酸序列可包括于任何表达载体中，只要载体在宿主中可复制和存活即可。***表达载体的多核苷酸序列受指导mRNA合成的适当启动子(例如LTR启动子)控制。该表达载体还包含用于起始翻译的核糖体结合位点、转录终止子、增强转录的增强子元件(例如SV40晚期增强子)、和将载体保持在宿主内的适当的选择性标记物(例如氨苄青霉素抗性基因)。宿主细胞可以是高等真核细胞例如哺乳动物细胞或昆虫细胞，低等真核细胞例如酵母，或原核细胞例如细菌。可采用多种方案将该构造物引入宿主细胞。本领域技术人员会明白上述所有步骤。克隆步骤以及宿主与载体的选择是本领域中已知的。(Sambrook等，Molecular Cloning：A Laboratory Manual，Second Edition，ColdSpring Harbor，N.Y.，(1989))。The polynucleotide sequence can be included in any expression vector so long as the vector is replicable and viable in the host. The polynucleotide sequence inserted into the expression vector is under the control of an appropriate promoter (eg, the LTR promoter) that directs mRNA synthesis. The expression vector also contains a ribosome binding site for initiating translation, a transcription terminator, an enhancer element to enhance transcription (eg, the SV40 late enhancer), and an appropriate selectable marker to maintain the vector within the host ( such as ampicillin resistance gene). Host cells can be higher eukaryotic cells such as mammalian cells or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacteria. The construct can be introduced into a host cell using a variety of protocols. All of the above steps will be understood by those skilled in the art. Cloning procedures and selection of hosts and vectors are known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989)).

可以在合适的启动子控制下在宿主细胞中合成蛋白质。还可将无细胞翻译***用来生产这类蛋白质，其中应用得自本发明的DNA构建物的RNAs。本发明的多肽也可用惯常的肽合成仪来合成。Proteins can be synthesized in host cells under the control of suitable promoters. Cell-free translation systems can also be used to produce such proteins, using RNAs derived from the DNA constructs of the present invention. The polypeptides of the present invention can also be synthesized using conventional peptide synthesizers.

在转化或转染适当的宿主株并使该宿主株生长至合适的细胞密度之后，利用适当方法诱导选择的启动子(例如改变温度或化学诱导)，再将细胞培养一段时间。一般通过离心作用收获细胞，用物理方法或化学法破裂后，运用各种方法(例如色谱法，HPLC)纯化至均匀。视采用的宿主细胞而定，该多肽可通过某种方法(例如糖基化)修饰。本发明的多肽还可包括一个初始蛋氨酸残基。After transformation or transfection of an appropriate host strain and growth of the host strain to an appropriate cell density, the promoter of choice is induced using an appropriate method (eg, temperature changes or chemical induction), and the cells are cultured for an additional period of time. Cells are typically harvested by centrifugation, disrupted physically or chemically, and purified to homogeneity using various methods (eg chromatography, HPLC). Depending on the host cell employed, the polypeptide can be modified by a method such as glycosylation. The polypeptides of the present invention may also include an initial methionine residue.

长形式Ckβ-8是白细胞的趋化物，因而可用于多种免疫调节作用和炎症作用以及一些疾病中。该蛋白质主要是在造血组织中表达的。Ckβ-8多肽对白细胞的一个重要生物效应是刺激Ca2+贮存物的迁移。这种迁移与细胞的功能性激活有关。已发现用包含4种短形式Ckβ-8(SEQ ID NO：1～4)的混合物刺激分化的EOL-3细胞会导致胞内Ca2+剂量依赖性地迅速增多。单独测试时，具有SEQ IDNO：4的短形式是活性最高的。反之，长形式Ckβ-8使Ca2+迁移的活性比短形式约小1000倍。本发明短形式的效能可能对于该趋化因子的治疗应用功效有意义，这些治疗应用包括但不限于：在化学疗法中保护骨髓干细胞以防化疗剂损伤，除去白血病细胞，刺激免疫响应，调节血细胞生成和淋巴细胞运输，治疗牛皮癣、固体肿瘤，增强宿主防御以抗慢性和急性感染，和刺激伤口愈合。The long form of Ck[beta]-8 is a chemoattractant for leukocytes and is therefore useful in a variety of immunomodulatory and inflammatory effects as well as in some diseases. This protein is mainly expressed in hematopoietic tissues. An important biological effect of Ckβ-8 polypeptides on leukocytes is the stimulation of migration of Ca2+ stores. This migration is associated with functional activation of cells. It has been found that stimulation of differentiated EOL-3 cells with a mixture comprising four short forms of Ckβ-8 (SEQ ID NOs: 1-4) results in a dose-dependent rapid increase in intracellular Ca2+. When tested alone, the short form with SEQ ID NO: 4 was the most active. Conversely, the long form of Ckβ-8 is approximately 1000-fold less active in Ca2+ transport than the short form. The efficacy of the short form of the invention may be of interest for the efficacy of the chemokine for therapeutic applications including, but not limited to, protecting bone marrow stem cells from damage by chemotherapeutic agents in chemotherapy, removing leukemia cells, stimulating immune responses, regulating blood cells Generation and lymphocyte trafficking, treatment of psoriasis, solid tumors, enhancement of host defenses against chronic and acute infections, and stimulation of wound healing.

本发明的多核苷酸和多肽可作为研究试剂用于DNA的合成和DNA载体的制备，用于开发治疗人类疾病的治疗剂和诊断剂(例如在未成熟造血祖细胞的扩充中)。该截短了的Ckβ-8多核苷酸序列的片段也可用作杂交探针以分离具有高度序列相似性的其它基因。本领域技术人员熟悉这类实验技术。The polynucleotides and polypeptides of the invention can be used as research reagents for the synthesis of DNA and the preparation of DNA vectors for the development of therapeutics and diagnostics for the treatment of human diseases (eg, in the expansion of immature hematopoietic progenitor cells). Fragments of this truncated Ckβ-8 polynucleotide sequence can also be used as hybridization probes to isolate other genes with high sequence similarity. Those skilled in the art are familiar with such experimental techniques.

本发明还涉及这些序列作为诊断分析的部分在检测疾病或检测对于与核酸序列中存在的突变相关的疾病的敏感性方面的应用。这类疾病与趋化因子多肽的表达不足有关。例如，携带Ckβ-8基因中突变的个体可通过如PCR的多种技术在DNA水平检测。基于DNA序列差异的基因检验可这样进行：存在或者不存在变性剂时，通过检测凝胶中DNA片段的电泳迁移率的改变。特异性DNA序列的检测例如可通过下列方法来实现：杂交、RNase保护、化学裂解、直接DNA测序、RFLP分析、和基因组DNA的DNA印迹法。突变也可通过原位分析检测。The present invention also relates to the use of these sequences as part of a diagnostic assay to detect disease or to detect susceptibility to disease associated with mutations present in nucleic acid sequences. Such diseases are associated with insufficient expression of chemokine polypeptides. For example, individuals carrying mutations in the Ckβ-8 gene can be detected at the DNA level by various techniques such as PCR. Genetic testing based on DNA sequence differences can be performed by detecting changes in the electrophoretic mobility of DNA fragments in the gel in the presence or absence of a denaturant. Detection of specific DNA sequences can be accomplished, for example, by hybridization, RNase protection, chemical cleavage, direct DNA sequencing, RFLP analysis, and Southern blotting of genomic DNA. Mutations can also be detected by in situ analysis.

本发明还涉及检测各种组织中Ckβ-8蛋白质变化量的诊断分析，因为与正常对照组织样品相比蛋白质的超量表达可检测疾病的存在或检测对于例如肿瘤的疾病的敏感性。本领域技术人员熟知用于检测得自宿主的样品中Ckβ-8蛋白质含量的分析法，这些分析法包括：放射免疫分析、竞争性结合分析、蛋白质印迹分析、ELISA分析和“夹心”分析。The present invention also relates to diagnostic assays that detect changes in Ckβ-8 protein in various tissues, since overexpression of the protein compared to normal control tissue samples can detect the presence of disease or detect sensitivity to disease such as tumors. Assays for the detection of Ckβ-8 protein content in samples obtained from a host are well known to those of skill in the art and include: radioimmunoassays, competitive binding assays, Western blot assays, ELISA assays and "sandwich" assays.

(A)电话：(610)270-5219(B)电传：(610)270-5090(2)SEQ ID NO：1的信息：(i)序列特征：(A)长度：82(B)类型：氨基酸(D)拓扑学：线型(xi)序列描述：SEQ ID NO：1GLU ASN PRO VAL LEU LEU ASP ARG PHE HIS ALA THR SER ALA ASP1               5                  10                  15CYS CYS ILE SER TYR THR PRO ARG SER ILE PRO CYS SER LEU LEU20                 25                  30GLU SER TYR PHE GLU THR ASN SER GLU CYS SER LYS PRO GLY VAL35                 40                  45ILE PHE LEU THR LYS LYS GLY ARG ARG PHE CYS ALA ASN PRO SER50                 55                  60ASP LYS GLN VAL GLN VAL CYS MET ARG MET LEU LYS LEU ASP THR65                 70                  75ARG ILE LYS THR ARG LYS ASN80(2)SEQ ID NO：2的信息：(i)序列特征：(A)长度：77(B)类型：氨基酸(D)拓扑学：线型(xi)序列描述：SEQ ID NO：2：(A) Tel: (610) 270-5219 (B) Fax: (610) 270-5090 (2) Information of SEQ ID NO: 1: (i) Sequence Features: (A) Length: 82 (B) Type : Amino Acid (D) Topology: Linear (XI) Sequence Description: SEQ ID NO: 1GLU ASN Pro Val Leu ASP Arg Phe His Ala Thr Ser Ala ASP1 5 10 15Cys Cys Ile Ser Tyr THR Pro Arg Ser ILEL PRO CYS SER Leu Leu20 25 30Glu Ser Tyr Phe Glu THR ASN Ser Glu Cys Ser Lys Pro Gly Val35 40 45ile Phe Leu THR LYS LYS GLY Arg Arg Phe Cys Ala Asn Pro Ser 50 55 60ASP Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu ASP THR65 70         75 ARG ILE LYS THR ARG LYS ASN80(2) Information of SEQ ID NO: 2: (i) Sequence Features: (A) Length: 77 (B) Type: Amino Acids (D) Topology: Linear (xi) Sequence Description : SEQ ID NO: 2:

本发明提供了鉴定趋化因子多肽的受体的方法。编码该受体的基因可通过本领域技术人员已知的多种方法来鉴定，这些方法包括配体淘选和FACS分选。The present invention provides methods of identifying receptors for chemokine polypeptides. The gene encoding the receptor can be identified by a variety of methods known to those of skill in the art, including ligand panning and FACS sorting.

用于受体鉴定的一个备选方法包括将标记的多肽与细胞膜或者表达该受体分子的提取物制剂进行光亲和性连接。可分离出标记的复合体并进行蛋白质微量测序分析。所得氨基酸序列将用于设计一组简并寡核苷酸探针以筛选鉴定编码推定受体的基因的cDNA文库。An alternative method for receptor identification involves photoaffinity linking of labeled polypeptides to cell membranes or to extract preparations expressing the receptor molecule. Labeled complexes can be isolated and analyzed by protein microsequencing. The resulting amino acid sequences will be used to design a panel of degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding putative receptors.

本发明提供了筛选化合物的方法，以鉴定本发明的趋化因子多肽的***和拮抗剂。可这样分析趋化性，即：将被这些多肽趋化的细胞置于孔径大到足以容纳细胞(5mm)的滤器上部。将可能的***或拮抗剂溶液置于底室，使得在一段时间后细胞通过膜迁移或者防止细胞通过膜迁移。也可将Ckβ-8的受体与标记的多肽在某化合物的存在下一起保温。可测定当不存在多肽时该化合物阻止该相互作用的能力或者与该受体相互作用的能力。The present invention provides methods of screening compounds to identify agonists and antagonists of the chemokine polypeptides of the present invention. Chemotaxis can be assayed by placing the cells chemotaxis by these polypeptides on top of a filter with a pore size large enough to accommodate the cells (5 mm). A solution of possible agonists or antagonists is placed in the bottom chamber to allow or prevent cell migration through the membrane after a period of time. The receptor for Ckβ-8 can also be incubated with the labeled polypeptide in the presence of a compound. The ability of the compound to prevent the interaction or to interact with the receptor in the absence of the polypeptide can be determined.

截短了的Ckβ-8的可能拮抗剂实例包括：抗体、与该多肽结合的寡核苷酸或者与野生型多肽的受体结合的多肽，但不能保持生物活性。用于控制基因表达的反义技术也可能是潜在的拮抗剂。对本领域技术人员而言，上述技术的可获得性是显而易见的。Examples of possible antagonists of truncated Ckβ-8 include antibodies, oligonucleotides that bind to the polypeptide, or polypeptides that bind to the receptor of the wild-type polypeptide, but do not retain biological activity. Antisense technologies used to control gene expression may also be potential antagonists. The availability of such techniques will be apparent to those skilled in the art.

拮抗剂可用于治疗例如下述传染病：矽肺、肉样瘤病、自发性肺纤维化、自发性嗜曙红细胞过多综合征和内毒素的休克-所有这些治疗应用都是通过防止产生本发明的多肽而进行的。该类拮抗剂也可用于通过防止动脉壁内单核细胞浸润而治疗粥样硬化。拮抗剂可用于治疗组胺介导的***反应和包括真皮的各种免疫病。Antagonists are useful in the treatment of infectious diseases such as silicosis, sarcoidosis, idiopathic pulmonary fibrosis, idiopathic hypereosinophilic syndrome and endotoxin shock - all of which are therapeutically useful by preventing the development of the present invention. of polypeptides. Such antagonists are also useful in the treatment of atherosclerosis by preventing the infiltration of mononuclear cells within the arterial wall. Antagonists are useful in the treatment of histamine-mediated allergy and various immune disorders including the dermis.

拮抗剂还可用于通过防止创伤区吸引单核细胞而治疗慢性和急性炎症。它们还可用于通过防止单核细胞流入感染区而治疗炎性肺病、一般的炎症和类风湿性关节炎。Antagonists can also be used to treat chronic and acute inflammation by preventing the attraction of monocytes to the wounded area. They can also be used to treat inflammatory lung disease, general inflammation and rheumatoid arthritis by preventing the influx of monocytes into the infected area.

拮抗剂也可用于治疗再生障碍性贫血或脊髓发育不良综合征中的骨髓缺乏症。它们还可用于治疗气喘和***反应以及上皮下的基膜纤维化，它是气喘肺的特征。Antagonists may also be used to treat bone marrow deficiency in aplastic anemia or myelodysplastic syndromes. They are also useful in the treatment of asthma and allergies as well as subepithelial basement membrane fibrosis, which is characteristic of asthmatic lungs.

趋化因子多肽以及***和拮抗剂可与例如下列的适当药物载体组合使用：盐水、缓冲盐水、葡萄糖、水、甘油、乙醇、及其组合。配方应适合施用方式。本发明还提供包括一个或多个贮器的药物包或试剂盒，在这些贮器中装有一种或多种本发明的药物组合物成分。多肽、***和拮抗剂可与其它治疗化合物结合使用。Chemokine polypeptides, as well as agonists and antagonists, can be used in combination with suitable pharmaceutical carriers such as saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of application. The present invention also provides pharmaceutical packs or kits comprising one or more receptacles in which are contained one or more components of the pharmaceutical composition of the present invention. Polypeptides, agonists and antagonists can be used in combination with other therapeutic compounds.

该药物组合物可按便利的方式施用，例如通过局部的、静脉内的、腹膜内的、肌内的、肿瘤内的、皮下的、鼻内的或真皮内的途径，用量应对于治疗特定的病症有疗效。本领域技术人员会明白这些应用。The pharmaceutical composition may be administered in a convenient manner, eg, by topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes, in an amount appropriate to the treatment-specific The disease is curative. Those skilled in the art will understand these applications.

趋化因子多肽，***或拮抗剂多肽，可按本发明通过活体内表达这类多肽而得以应用。这种基因疗法是本领域中熟知的。例如，患者的细胞可用编码短形式Ckβ-8多肽的DNA或RNA进行来自体内的工程化处理，再将表达这些多肽的工程化细胞提供给患者。类似地，可对细胞进行活体内工程化处理以在活体内表达多肽。Chemokine polypeptides, agonist or antagonist polypeptides, can be used in accordance with the present invention by expressing such polypeptides in vivo. Such gene therapy is well known in the art. For example, a patient's cells can be engineered ex vivo with DNA or RNA encoding short forms of Ckβ-8 polypeptides, and engineered cells expressing these polypeptides can be provided to the patient. Similarly, cells can be engineered in vivo to express polypeptides in vivo.

正如本领域中已知那样，可应用包含编码本发明的多肽的RNA的逆转录病毒颗粒。应用逆转录病毒质粒载体通过各种方法(例如电穿孔法)转导包装细胞系(例如PE501)而形成生产者细胞系。在一个优选的实施方案中，包含多核苷酸序列的逆转录病毒表达载体，受例如LTR的启动子控制并含有一个可选择的药物抗性标记物(例如neo)。这些载体的制备和产生的细胞系应是本领域技术人员熟悉的，并且可采用本文包括的技术。生产者细胞系产生感染性逆转录病毒载体颗粒，该颗粒包括编码该多肽的核酸序列。然后可将这类逆转录病毒载体颗粒用于在体外或者在活体内转导真核细胞，例如成纤维细胞或内皮细胞。然后转导的真核细胞就表达编码该蛋白质的核酸序列。Retroviral particles comprising RNA encoding the polypeptides of the invention may be employed as known in the art. Producer cell lines are formed using retroviral plasmid vectors to transduce packaging cell lines (eg, PE501) by various methods (eg, electroporation). In a preferred embodiment, the retroviral expression vector comprising the polynucleotide sequence is controlled by a promoter such as LTR and contains a selectable drug resistance marker (eg neo). The preparation of these vectors and the resulting cell lines should be familiar to those of skill in the art and the techniques included herein can be employed. The producer cell line produces infectious retroviral vector particles that include the nucleic acid sequence encoding the polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells, such as fibroblasts or endothelial cells, in vitro or in vivo. The transduced eukaryotic cells then express the nucleic acid sequence encoding the protein.

本发明的序列对于染色体鉴定也有价值。这些序列被特异性地导向至单个染色体上特定的部位并与之杂交。DNA对染色体作图是关联与疾病相关的基因中一个重要步骤。本领域技术人员已知的一种技术包括制备用于体细胞杂种的PCR筛选的引物，其中的体细胞杂种包含各个人染色体。只有那些包含与该引物相应的人基因的杂种才会产生扩增的片段。本领域技术人员已知的其它作图方法包括但不限于：原位杂交，用标记的流式分选染色体预筛选，应用相同的PCR引物亚定位(subloealization)至特定染色体的片段上，和通过杂交预选以构建染色体特异性cDNA文库。cDNA克隆与中期染色体扩散的荧光原位杂交(FISH)可用于在一步中提供准确的染色***置。The sequences of the invention are also valuable for chromosomal identification. These sequences are specifically targeted to and hybridize to specific sites on individual chromosomes. Mapping of DNA to chromosomes is an important step in associating genes associated with disease. One technique known to those of skill in the art involves the preparation of primers for PCR screening of somatic cell hybrids comprising individual human chromosomes. Only those hybrids containing the human gene corresponding to this primer will produce amplified fragments. Other mapping methods known to those of skill in the art include, but are not limited to, in situ hybridization, pre-screening of chromosomes with labeled flow sorting, application of the same PCR primers for subloealization to fragments of a particular chromosome, and Hybridization preselection to construct chromosome-specific cDNA libraries. Fluorescence in situ hybridization (FISH) of cDNA clones with metaphase chromosomal spread can be used to provide accurate chromosomal locations in one step.

一旦某序列已被绘至准确的染色***置，则该序列的物理位置会与遗传学图数据相关联。基因和疾病之间的关系可通过连锁分析鉴定。感染的和未感染的个体之间在cDNA或基因组序列方面的任何差异可预示疾病。例如，只在感染的个体中发现的DNA中突变可能是疾病的病因。Once a sequence has been mapped to the exact chromosomal location, the physical location of the sequence is associated with the genetic map data. Relationships between genes and diseases can be identified by linkage analysis. Any differences in cDNA or genomic sequence between infected and uninfected individuals can be predictive of disease. For example, mutations in DNA found only in infected individuals may be the cause of disease.

多肽，它们的片段或其它衍生物，或其类似物，或者表达它们的细胞又可作为免疫原产生抗体。这些抗体例如可以是多克隆或单克隆抗体。本发明还包括嵌合的、单链的和人源化抗体，以及Fab片段，或者Fab表达文库的产物。本领域中已知的各种方法可用于产生这类抗体和片段。通过标准方法制备的多克隆抗体可用于从表达该多肽的组织分离该多肽。为制备单克隆抗体，可应用提供通过连续传代细胞系培养物生产的抗体的任何技术。所述生产单链抗体的技术可适用于生产抗本发明的免疫原性多肽的单链抗体。转基因小鼠也可用于表达抗本发明的免疫原性多肽的人源化抗体。Polypeptides, fragments or other derivatives thereof, or analogs thereof, or cells expressing them may in turn serve as immunogens for the production of antibodies. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or products of Fab expression libraries. Various methods known in the art can be used to generate such antibodies and fragments. Polyclonal antibodies prepared by standard methods can be used to isolate the polypeptide from tissue expressing the polypeptide. To prepare monoclonal antibodies, any technique that provides antibodies produced by serially passaged cell line cultures can be applied. The techniques for producing single chain antibodies can be applied to produce single chain antibodies against the immunogenic polypeptides of the invention. Transgenic mice can also be used to express humanized antibodies against the immunogenic polypeptides of the invention.

本发明通过下述实施例将得以进一步阐述；但是，不能认为本发明受这些实施例的限制。为了便于理解下述实施例，下面将简述一些常见方法和/或术语。The present invention will be further illustrated by the following examples; however, the present invention should not be considered limited by these examples. To facilitate understanding of the following embodiments, some common methods and/or terminology will be briefly described below.

本文的起始质粒要么是可商购的，在未限制的基础上可公开获得，或者可按发表的方法从可获得的质粒构建。此外，普通技术人员会明白本领域中与所述相当的质粒。本文应用的用于“消化”DNA的各种限制酶是可商购的，并且它们的反应条件、所用的辅因子和其它要求是普通技术人员已知的。应用8％聚丙烯酰胺凝胶进行按大小分离裂解的片段。The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by published methods. In addition, one of ordinary skill will recognize plasmids equivalent to those described in the art. Various restriction enzymes used herein for "digesting" DNA are commercially available, and their reaction conditions, cofactors used and other requirements are known to those of ordinary skill. The cleaved fragments were separated by size using an 8% polyacrylamide gel.

寡核苷酸表示单链的多脱氧核苷酸或可用化学法合成的两个互补的多脱氧核苷酸链。这种合成的寡核苷核没有5′磷酸，于是如果不加入磷酸和ATP就不能在激酶存在下与另一个寡核苷酸连接。合成的寡核苷酸将与未脱磷酸的片段连接。除非另有叙述，是用Graham，F.和Van der Eb，A.在Virology 1973，52，456-457中描述的方法进行转化的。Oligonucleotide means a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotide cores have no 5' phosphate and thus cannot be linked to another oligonucleotide in the presence of a kinase without the addition of phosphate and ATP. Synthetic oligonucleotides will be ligated to fragments that are not dephosphorylated. Unless otherwise stated, transformation was performed using the methods described by Graham, F. and Van der Eb, A. in Virology 1973, 52, 456-457.

下述实施例只是为了阐述而不想限制本发明。实施例实施例1：Ckβ-8的细菌表达和纯化先用PCR引物扩增编码Ckβ-8的DNA序列，ATCC#75676，该PCR引物相应于加工过的Ckβ-8蛋白质(减去信号肽序列)的5′和3＇端序列和Ckβ-8基因的3′载体序列。5′寡核苷酸引物的序列为5′TCAGGATCCGTCACAAAAGATGCAGA3′(SEQ ID NO：5)，而3′引物的序列为5′CGCTCTAGAGTAAAACGACGGCCAGT3′(SEQID NO：6)。这些引物分别包含BamHI和XbaI限制酶切位点，这些位点用于克隆入氨苄青霉素抗性细菌表达载体PQE-9(Qiagen，Inc.，Chatsworth，CA)的多接头区。已扩增的序列与编码组氨酸标记物和核糖体结合位点(RBS)的序列一起连接到读框中的PQE-9中。该连接反应被用于转化大肠杆菌株M15/rep4(Qiagen)，所述菌株包含质粒pREP4的多个复制物。pREP4表达lacI阻抑物并且还赋予卡那霉素抗性。根据它们在用氨苄青霉素/卡那霉素补充的LB板上生长的能力来选择转化体。包含所需构建物的克隆(分离并用限制酶切分析确证)在液体培养物中生长一夜，该培养物在用Amp(100μg/ml)和Kan(25μg/ml)补充的LB培养基中。将该培养物用于在1∶100～1∶250的比例下接种大量的培养物。使细胞生长至光密度600为0.4～0.6之间。然后添加IPTG(异丙基-B-D-硫代半乳糖-吡喃糖苷)至最终浓度为1mM。再使细胞生长3～4小时，然后通过离心收集。将细胞沉淀溶于离液剂6M盐酸胍中，澄清，在Nickel-Chelate柱上进行色谱分离，其中分离条件应使之通过含有6-His标记物的蛋白质而紧密结合(Hochuli，E.等J.Chromatography 1984，411，177-184)。用6M盐酸胍pH5从柱上洗脱截短了的Ckβ-8(纯度为95％)，并且为了再生而调节至3M盐酸胍，100mM磷酸钠，10mM谷胱甘肽(还原态)和2mM谷胱甘肽(氧化态)。在该溶液中培养12小时后，将该蛋白质在10mM磷酸钠中透析。实施例2：重组Ckβ-8在COS细胞中的表达表达质粒CMV-Ckβ-8HA得自载体pcDNAI/Amp(Invitrogen)，该载体含氨苄青霉素抗性基因和CMV启动子，接着是多接头区、SV40内含子和聚腺苷酸化位点。一个编码截短了的Ckβ-8序列和在读框中与3′末端融合的HA标记物的DNA片段被克隆入该载体的多接头区；因此，在CMV启动子作用下指导了重组蛋白质的表达。如前所述(I.Wilson等，Cell 1984，37：767)，该HA标记物相应于得自流感血凝素蛋白质的表位。HA标记物与靶蛋白质的融合能容易地采用识别HA表位的抗体来检测该重组蛋白质。The following examples are for illustration only and are not intended to limit the invention. EXAMPLES Example 1: Bacterial expression and purification of Ckβ-8 The DNA sequence encoding Ckβ-8 was first amplified with PCR primers, ATCC #75676, corresponding to the processed Ckβ-8 protein (minus the signal peptide sequence ) of the 5' and 3' end sequences and the 3' vector sequence of the Ckβ-8 gene. The sequence of the 5' oligonucleotide primer was 5'TCAGGATCCGTCACAAAAGATGCAGA3' (SEQ ID NO:5) and the sequence of the 3' primer was 5'CGCTCTAGAGTAAAACGACGGCCAGT3' (SEQ ID NO:6). These primers contained BamHI and XbaI restriction sites, respectively, which were used for cloning into the polylinker region of the ampicillin-resistant bacterial expression vector PQE-9 (Qiagen, Inc., Chatsworth, CA). The amplified sequence was ligated into PQE-9 in frame with sequences encoding the histidine tag and ribosome binding site (RBS). This ligation reaction was used to transform E. coli strain M15/rep4 (Qiagen), which contains multiple copies of plasmid pREP4. pREP4 expresses the lacI repressor and also confers kanamycin resistance. Transformants were selected based on their ability to grow on LB plates supplemented with ampicillin/kanamycin. Clones containing the desired construct (isolated and confirmed by restriction analysis) were grown overnight in liquid culture in LB medium supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). This culture was used to inoculate a large number of cultures at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 between 0.4 and 0.6. IPTG (isopropyl-B-D-thiogalactopyranoside) was then added to a final concentration of 1 mM. Cells were grown for an additional 3-4 hours and then collected by centrifugation. The cell pellet was dissolved in the chaotropic agent 6M guanidine hydrochloride, clarified, and chromatographed on a Nickel-Chelate column under conditions such that it was tightly bound by proteins containing the 6-His tag (Hochuli, E. et al. J. . Chromatography 1984, 411, 177-184). Truncated Ckβ-8 (95% purity) was eluted from the column with 6M guanidine HCl pH5 and adjusted for regeneration to 3M guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (reduced) and 2 mM glutathione glutathione (oxidized state). After 12 hours of incubation in this solution, the protein was dialyzed against 10 mM sodium phosphate. Example 2: Expression of recombinant Ckβ-8 in COS cells The expression plasmid CMV-Ckβ-8HA was obtained from the vector pcDNAI/Amp (Invitrogen), which contained the ampicillin resistance gene and the CMV promoter, followed by the polylinker region, SV40 intron and polyadenylation site. A DNA fragment encoding a truncated Ckβ-8 sequence and an HA tag fused in-frame to the 3' end was cloned into the polylinker region of this vector; thus, directed expression of the recombinant protein under the CMV promoter . As previously described (I. Wilson et al., Cell 1984, 37:767), this HA marker corresponds to an epitope derived from the influenza hemagglutinin protein. Fusion of the HA tag to the target protein allows the recombinant protein to be readily detected using antibodies that recognize the HA epitope.

为了表达该重组短形式的Ckβ-8，应用表达载体通过DEAE-葡聚糖方法(J.Sambrook，E.Fritsch，T.Maniatis，Molecular Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory Press，(1989))转染COS细胞。转染2天后用35S-半胱氨酸将细胞标记8小时。然后收集培养基并用洗涤剂(RIPA缓冲剂(150mM NaCl、1％NP-40，0.1％SDS，1％NP-40，0.5％DOC，50mM Tris，pH7.5))(Wilson，I.等，ID.1984，37：767)裂解细胞。用HA特异性单克隆抗体使细胞裂解物和培养基沉淀。将沉淀的蛋白质在15％SDS-PAGE凝胶上分析。实施例3：应用杆状病毒表达***表达和纯化短形式的趋化因子Ckβ-8用设计成表达Ckβ-8cDNA的重组杆状病毒感染SF9细胞。在MOI为2时于10升培养基中感染细胞，并在低血清条件下于28℃时培养72-96小时。通过低速离心除去已感染培养物中的细胞碎片。往上清液中加入蛋白酶抑制剂混合物至最终浓度为20μg/ml(1μg/ml亮抑酶肽，1μ/ml E-64和1mM EDTA)。这些特定的培养条件(即低血清)产生数种NH2端截短形式的Ckβ-8多肽。通过将20-30μl上清液装入15％SDS-PAGE凝胶而监控上清液中Ckβ-8的含量。以可见谱带形式检测到截短形式的Ckβ-8，相当于每升数mg的表达量。截短的Ckβ-8通过如下三步纯化法进一步纯化：1)肝素结合亲和色谱。将杆状病毒培养物的上清液与1/3体积、含有100mMHEPES/MEM/NaOAc pH6的缓冲液混合，并滤过0.22μm膜。然后将样品加到肝素结合柱(HE1 poros 20，BIO-Perceptive System Inc.)上。在约300mM NaCl时，于50～500mM NaCl在50mMHEPES/MES/NaOAc pH6的线性梯度中洗脱短形式Ckβ-8；2)阳离子交换色谱。用含有50mM HEPES/MES/NaOAc pH6的缓冲液将肝素色谱法浓缩后的蛋白质稀释5倍。再将所得混合物加入阳离子交换柱(S/M poros 20，BIO-Perceptive System Inc.)。在250mM NaCl时，于25～300mM NaCl在50mM HEPES/MES/NaOAc pH6的线性梯度中洗脱截短了的Ckβ-8；3)大小排斥色谱。在阳离子交换色谱之后，截短了的Ckβ-8通过加入大小排斥柱(HW50，TOSO HAAS，1.4x45cm)而被进一步纯化。对纯化后的样品进行氨基酸测序，表明该样品是至少4种主要序列的混合物，这4种序列对应于4种截短形式的蛋白质(SEQ ID NO：1、2、3和4)。实施例4：用包含短形式Ckβ-8的混合物刺激EOL-3细胞和PBLsEOL-3细胞在标准生长条件下生长和用1μM丁酸钠分化两周。PBLs(外周血白细胞)通过静脉穿刺由人供血者得到。用标准方法纯化PBLs。这两种细胞制剂分别用FURA-2(1μM)荷载45分钟。在1x106/ml(总量为2ml)下将细胞转入塑料比色池。在加入浓度为0.33～33nM的短形式Ckβ-8(871a、871b、871c、871RP)或长形式Ckβ-8(889)之前，将荧光分光光度计调节至基线记录状态。用该分光光度计监测荧光的增强。这样计算细胞内游离Ca2+的变化：先用Triton x100在过量的Ca2+存在下裂解细胞(得到Fmax)，然后加入5mM EGTA而得Fmin。根据极小值和极大值可算出游离Ca2+的比率。MCP-1(单核细胞趋化性蛋白-1)是已知的趋化因子，并用作阳性对照。To express this recombinant short form of Ckβ-8, an expression vector was used by the DEAE-dextran method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1989) ) transfected COS cells. Cells were labeled with 35S-cysteine for 8 hours 2 days after transfection. The medium was then collected and washed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)) (Wilson, I. et al., ID. 1984, 37:767) Lysate cells. Cell lysates and media were pelleted with HA-specific monoclonal antibodies. The precipitated proteins were analyzed on 15% SDS-PAGE gels. Example 3: Expression and Purification of Short Form Chemokine Ckβ-8 Using Baculovirus Expression System SF9 cells were infected with recombinant baculovirus designed to express Ckβ-8 cDNA. Cells were infected in 10 liters of medium at an MOI of 2 and incubated for 72-96 hours at 28°C under low serum conditions. Cell debris from the infected culture was removed by low speed centrifugation. The protease inhibitor cocktail was added to the supernatant to a final concentration of 20 μg/ml (1 μg/ml leupeptin, 1 μ/ml E-64 and 1 mM EDTA). These specific culture conditions (ie, low serum) produced several NH2-terminally truncated forms of the Ck[beta]-8 polypeptide. The content of Ckβ-8 in the supernatant was monitored by loading 20-30 μl of the supernatant on a 15% SDS-PAGE gel. A truncated form of Ckβ-8 was detected as a visible band, corresponding to an expression of several mg per liter. The truncated Ckβ-8 was further purified by the following three-step purification method: 1) Heparin-binding affinity chromatography. The supernatant of the baculovirus culture was mixed with 1/3 volume of buffer containing 100 mM HEPES/MEM/NaOAc pH 6 and filtered through a 0.22 μm membrane. The sample was then applied to a heparin-binding column (HE1 poros 20, BIO-Perceptive System Inc.). The short form Ckβ-8 was eluted in a linear gradient of 50-500 mM NaCl in 50 mM HEPES/MES/NaOAc pH 6 at about 300 mM NaCl; 2) Cation exchange chromatography. Heparin chromatography-concentrated proteins were diluted 5-fold with a buffer containing 50 mM HEPES/MES/NaOAc pH 6. The resulting mixture was then applied to a cation exchange column (S/M poros 20, BIO-Perceptive System Inc.). Truncated Ckβ-8 was eluted in a linear gradient of 25-300 mM NaCl in 50 mM HEPES/MES/NaOAc pH 6 at 250 mM NaCl; 3) Size exclusion chromatography. After cation exchange chromatography, truncated Ckβ-8 was further purified by addition of a size exclusion column (HW50, TOSO HAAS, 1.4x45 cm). Amino acid sequencing of the purified sample indicated that the sample was a mixture of at least 4 major sequences corresponding to 4 truncated forms of the protein (SEQ ID NOs: 1, 2, 3 and 4). Example 4: EOL-3 cells and PBLs EOL-3 cells were stimulated with a mixture containing the short form of Ck[beta]-8 to grow under standard growth conditions and differentiated with 1 [mu]M sodium butyrate for two weeks. PBLs (peripheral blood leukocytes) were obtained from human blood donors by venipuncture. PBLs were purified by standard methods. The two cell preparations were each loaded with FURA-2 (1 μM) for 45 minutes. Cells were transferred into plastic cuvette at 1x106/ml (2ml total). The spectrofluorometer was adjusted to baseline recording prior to addition of either the short form of Ckβ-8 (871a, 871b, 871c, 871RP) or the long form of Ckβ-8 (889) at concentrations of 0.33-33 nM. The enhancement of fluorescence was monitored with the spectrophotometer. Changes in free intracellular Ca2+ were calculated by first lysing cells with Triton x100 in the presence of excess Ca2+ (to obtain Fmax), then adding 5 mM EGTA to obtain Fmin. The ratio of free Ca2+ can be calculated from the minimum and maximum values. MCP-1 (monocyte chemotactic protein-1) is a known chemokine and was used as a positive control.

序列表(1)一般信息：(i)申请人：John White，Edward Appelbaum，DanielO′Shannessy，James Allan Fornwald和Kevin O′Donnell(ii)发明名称：趋化因子β-8的短形式(iii)序列数：6个(iV)地址：(A)通信地址：(B)街名：709 Swedeland Road(C)城市：King of Prussia(D)州名：PA(E)国家：美国(F)邮编：19406(V)计算机可读形式：(A)介质类型：磁盘，3.5英寸，1.44Mb容量(B)计算机：IBM486(C)操作***：WINDOWS FOR WORKGROUPS(D)软件：WORD PERFECT 5.1(vi)目前申请信息(A)申请号：尚未定(B)申请日：与此同时(C)分类：(Vii)在先申请信息：(A)申请号：(B)申请日：(viii)律师/代理人信息：(A)姓名：William T.Han(B)登记号：34，344(C)参考/档案号：P50381(ix)电信信息：Sequence Listing (1) General Information: (i) Applicants: John White, Edward Appelbaum, Daniel O'Shannessy, James Allan Fornwald and Kevin O'Donnell (ii) Invention Title: Short Form of Chemokine Beta-8 (iii) Serial Number: 6 (iV) Address: (A) Mailing Address: (B) Street Name: 709 Sweden Road (C) City: King of Prussia (D) State Name: PA (E) Country: United States (F) ZIP Code : 19406 (V) Computer readable form: (A) Media type: Disk, 3.5 inches, 1.44Mb capacity (B) Computer: IBM486 (C) Operating system: WINDOWS FOR WORKGROUPS (D) Software: WORD PERFECT 5.1 (vi) Current application information (A) Application number: Not yet determined (B) Application date: At the same time (C) Classification: (Vii) Prior application information: (A) Application number: (B) Application date: (viii) Attorney/ Agent Information: (A) Name: William T. Han (B) Registration Number: 34,344 (C) Reference/File Number: P50381(ix) Telecommunications Information:

LEU ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR1               5                  10                  15THR PRO ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU20                 25                  30THR ASN SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS35                 40                  45LYS GLY ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GLN50                 55                  60VAL CYS MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG65                 70                  75LYS ASN(2)SEQ ID NO：3的信息：(i)序列特征：(A)长度：76(B)类型：氨基酸(D)拓扑学：线型(xi)序列描述：SEQ ID NO：3：ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR1               5                  10                  15PRO ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR20                 25                  30ASN SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS LYS35                 40                  45GLY ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GLN VAL50                 55                  60CYS MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG LYS65                 70                  75ASN(2)SEQ ID NO：4的信息：(i)序列特征：(A)长度：75Leu ASP Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr1 5 10 15thr Pro Arg Ser ILEL PRHR PHE Glu20 25 30thr ASN Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu THR LYS35 40 45LYS Gly Arg Arg Phe Cys Ala Asn Pro Ser ASLYS GLN VAL GLN50 55 60VAL CYS MET Arg Met Leu Lys Leu ASP THR Arg Ile Lys THR ARG65 70 75LYS ASN (2) SEQ ID NO: 3 Information: (i) Sequence Features: (A) Length: 76 (B) Type: Amino Acids (D) Topology: Linear (xi) Sequence Description: SEQ ID NO: 3: ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR1 5 5 PRO 10 Arg Ser ILE Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu THR20 25 30ASN Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu THR LYS LYS35 40 45GLY Arg Arg Phe Cys Ala Asn Pro Ser Ass Lys Gln Val Gln Val50 55 60CYS MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG LYS65 70

(B)类型：氨基酸(D)拓扑学：线型(xi)序列描述：SEQ ID NO：4：ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR PRO1                5                  10                  15ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR ASN20                 25                  30SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS LYS GLY35                 40                  45ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GKN VAL CYS50                 55                  60MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG LYS ASN65                 70                  75(2)SEQ ID NO：5的信息：(i)序列特征：(A)长度：26(B)类型：核酸(C)链型：单链(D)拓扑学：线型(iv)反义：无(xi)序列描述：SEQ ID NO：5：TCAGGATCCG TCACAAAAGA TGCAGA    26(2)SEQ ID NO：6的信息：(i)序列特征：(A)长度：26(B)类型：核酸(C)链型：单链(D)拓扑学：线型(iv)反义：无(xi)序列描述：SEQ ID NO：6：CGCTCTAGAG TAAAACGACG GCCAGT    26(B) Type: Amino Acid (D) Topology: Linear (xi) Sequence Description: SEQ ID NO: 4: ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR PRO1 5 5 10 SER I PRO 1 5 AR G SER Y Leu Glu Ser Tyr Phe Glu THR ASN20 25 30Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu THR LYS LYS GLY35 40 45Arg Arg Phe Cys Ala Asn Val Cys 50 55 60Met Arg Met Leu Lys Leu ASP THR Arg ILE LYS THR ARG LYS ASN65 70 75(2) Information of SEQ ID NO: 5: (i) Sequence Features: (A) Length: 26 (B) Type: Nucleic Acid (C) Strand Type: Single Strand (D) Topology : Linear (iv) Antisense: None (xi) Sequence Description: SEQ ID NO: 5: TCAGGATCCG TCACAAAAGA TGCAGA 26 (2) Information of SEQ ID NO: 6: (i) Sequence Features: (A) Length: 26 ( B) Type: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear (iv) Antisense: None (xi) Sequence Description: SEQ ID NO: 6: CGCTCTAGAG TAAAACGACG GCCAGT 26

Claims (15)

1.包括SEQ ID NO：1的多肽。1. A polypeptide comprising SEQ ID NO:1. 2.包括SEQ ID NO：2的多肽。2. A polypeptide comprising SEQ ID NO:2. 3.包括SEQ ID NO：3的多肽。3. A polypeptide comprising SEQ ID NO:3. 4.包括SEQ ID NO：4的多肽。4. A polypeptide comprising SEQ ID NO:4. 5.包括权利要求1、2、3和4的多肽的混合物的组合物。5. A composition comprising a mixture of the polypeptides of claims 1, 2, 3 and 4. 6.包括编码权利要求1、2、3或4的多肽的多核苷酸的载体。6. A vector comprising a polynucleotide encoding the polypeptide of claim 1, 2, 3 or 4. 7.用权利要求6的载体进行工程化的宿主细胞。7. A host cell engineered with the vector of claim 6. 8.生产权利要求1、2、3或4的多肽的方法，该方法包括从宿主细胞表达该多肽，所述宿主细胞用包括编码权利要求1、2、3或4的多肽的多核苷酸的载体进行过工程化。8. A method of producing the polypeptide of claim 1, 2, 3 or 4, the method comprising expressing the polypeptide from a host cell using a polynucleotide comprising a polynucleotide encoding the polypeptide of claim 1, 2, 3 or 4 The vector is engineered. 9.权利要求1、2、3或4的多肽的***。9. An agonist of the polypeptide of claim 1, 2, 3 or 4. 10.权利要求1、2、3或4的多肽的拮抗剂。10. An antagonist of the polypeptide of claim 1, 2, 3 or 4. 11.治疗需要Ckβ-8的患者的方法，该方法包括给患者施用有效量的权利要求1、2、3或4的多肽。11. A method of treating a patient in need of Ck[beta]-8, the method comprising administering to the patient an effective amount of the polypeptide of claim 1, 2, 3 or 4. 12.治疗需要Ckβ-8的患者的方法，该方法包括给患者施用有效量的权利要求5的组合物。12. A method of treating a patient in need of Ck[beta]-8, the method comprising administering to the patient an effective amount of the composition of claim 5. 13.治疗需要抑制Ckβ-8的患者的方法，该方法包括给患者施用有效量的权利要求10的拮抗剂。13. A method of treating a patient in need of inhibition of Ck[beta]-8, the method comprising administering to the patient an effective amount of the antagonist of claim 10. 14.鉴定权利要求1、2、3或4的多肽的拮抗剂和***的方法，该方法包括：将选择的试验细胞，待筛选的化合物和权利要求1、2、3或4的多肽结合；并基于试验细胞的响应确定该化合物是否是有效的***或拮抗剂。14. The method for identifying the antagonist and the agonist of the polypeptide of claim 1, 2, 3 or 4, the method comprising: the selected test cell, the compound to be screened and the polypeptide of claim 1, 2, 3 or 4 are combined and determine whether the compound is a potent agonist or antagonist based on the response of the test cells. 15.诊断疾病或者对于与权利要求1、2、3或4的多肽表达不足相关的疾病的敏感性的方法，该方法包括确定编码该多肽的核酸序列中是否存在突变。15. A method of diagnosing a disease or susceptibility to a disease associated with insufficient expression of a polypeptide of claim 1, 2, 3 or 4, the method comprising determining the presence or absence of a mutation in a nucleic acid sequence encoding the polypeptide.