WO2002053590A1 - A novel fibrinolytic enzyme the polynucleotide encoding said polypeptide and the use thereof - Google Patents

Abstract

The invention discloses a new kind of fibrinolytic enzyme - fibrinolytic enzyme Z from earthworm, polynucleotide encoding said fibrinolytic enzyme Z and a process for producing said fibrinolytic enzyme Z by recombinant technology. It also discloses the method of applying the fibrinolytic enzyme Z for the treatment of various kinds of diseases, such as thrombo-embolia disease. In addition, the medicines composition which contains earthworm fibrinolytic enzyme Z is also disclosed.

Description

新的纤溶酶、 其编码序列及用途 技术领域  Novel plasmin, its coding sequence and use

本发明属于生物技术和医学领域， 具体地说， 本发明涉及新的编码纤溶酶-蚯 蚓纤溶酶 Z 的多核苷酸， 以及此多核苷酸编码的蛋白质。 本发明还涉及此多核苷 酸和蛋白质的制法和用途， 以及含该纤溶酶的药物组合物。 背景技术  The present invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a novel polynucleotide encoding plasmin-earthworm plasmin Z and a protein encoded by the polynucleotide. The invention also relates to a method for preparing and using the polynucleotide and protein, and a pharmaceutical composition containing the plasmin. Background technique

蚯蚓纤溶酶是从蚯蚓中提取到的一类能水解血纤维蛋白（原）的酶类。 它存在 于蚯蚓的消化道内， 分子量从 15, 000到 60， 000道尔顿。 蚯蚓纤溶酶不是单一的 一种酶， 而是具有相同功能的多种蛋白水解酶的总称。  Earthworm fibrinolytic enzymes are a class of enzymes that can hydrolyze fibrin (pro) from earthworms. It is found in the digestive tract of earthworms and has a molecular weight from 15,000 to 60,000 daltons. Earthworm fibrinolytic enzymes are not a single enzyme, but a collective term for multiple proteolytic enzymes with the same function.

研究结果表明蚯蚓纤溶酶具有双重功能， 除了能直接水解纤维蛋白外， 还能 激活血纤维蛋白溶酶原成血纤维蛋白溶酶， 从而具有间接水解纤维蛋白的作用。 体外药理实验的结果指出， 蚯蚓纤溶酶除了能直接溶解血块外， 还能刺激血管内 皮细胞释放 tPA， 从而增强体内的纤溶作用。 体内及体外血栓形成模型实验及人 工血栓溶解实验结果均指出， 蚯蚓纤溶酶具有明显的抗栓和溶栓作用。  The research results show that earthworm fibrinolytic enzymes have dual functions. In addition to directly hydrolyzing fibrin, they can also activate plasminogen into fibrinolytic enzymes, thus indirectly hydrolyzing fibrin. The results of in vitro pharmacological experiments indicate that, in addition to directly dissolving blood clots, earthworm fibrinolytic enzymes can stimulate the release of tPA from vascular endothelial cells, thereby enhancing fibrinolysis in vivo. The results of in vivo and in vitro thrombosis model experiments and artificial thrombolysis experiments indicate that earthworm fibrinolytic enzyme has obvious antithrombotic and thrombolytic effects.

基于蚯蚓纤溶酶这样一种特性， 国内外已将它制成口服胶囊用于临床上治疗 血栓栓塞性疾病。 例如， 商品名为龙心、 蚓激酶、 博洛克、 普恩复及溶栓胶囊等 药物都是以蚯蚓纤溶酶作为原料制成的。 但至今蚯蚓纤溶酶的结构还鲜为人知， 而且没有报道过任何一种具体蚯蚓纤溶酶的氨基酸序列或核苷酸序列。  Based on the characteristics of earthworm fibrinolytic enzyme, it has been made into oral capsules at home and abroad for clinical treatment of thromboembolic diseases. For example, medicines such as Longxin, lumbrokinase, Brock, Puenfu, and thrombolytic capsules are all made from earthworm fibrinolytic enzyme. However, the structure of earthworm fibrinolytic enzymes is still unknown, and the amino acid sequence or nucleotide sequence of any specific earthworm fibrinolytic enzyme has not been reported.

鉴于纤溶酶的在治疗血栓栓塞性疾病等方面具有巨大的应用前景， 因此本领 域迫切需要开发新的纤溶酶。 发明概述  In view of the huge application prospects of plasmin in the treatment of thromboembolic diseases, it is urgent to develop new plasmin in this field. Summary of invention

本发明的目的是提供一种新的纤溶酶蚯蚓纤溶酶以及其片段、 类似物和衍生 物。  An object of the present invention is to provide a novel plasmin, earthworm plasmin, and fragments, analogs and derivatives thereof.

本发明的另一目的是提供编码这些蛋白质的多核苷酸。  Another object of the invention is to provide polynucleotides encoding these proteins.

本发明的另一目的是提供生产这些蛋白质的方法以及该蛋白质和编码序列的 用途。 在本发明的第一方面， 提供新颖的分离出的蚯蚓纤溶酶 Z蛋白质， 该蛋白质 来自蚯蚓， 它包含： 具有 SEQ ID NO: 2氨基酸序列的蛋白质、 或其保守性变异蛋 白质、 或其活性片段、 或其活性衍生物。 较佳地， 该蛋白质是具有 SEQ ID NO: 2 氨基酸序列的蛋白质。 Another object of the present invention is to provide a method for producing these proteins and the use of the proteins and coding sequences. In a first aspect of the present invention, a novel isolated earthworm fibrinolytic enzyme Z protein is provided. The protein is derived from earthworms and comprises: a protein having the amino acid sequence of SEQ ID NO: 2, or a conservative variant protein thereof, or its activity Fragment, or an active derivative thereof. Preferably, the protein has SEQ ID NO: 2 Amino acid sequence of protein.

在本发明的第二方面， 提供编码分离的这些蛋白质的多核苷酸， 该多核苷酸 包含一核苷酸序列， 该核苷酸序列与选自下组的一种核苷酸序列有至少 70%同源 性： （a)编码上述蚯蚓纤溶酶 Z蛋白质的多核苷酸； 和 (b)与多核苷酸 (a)互补的多核 苷酸。 较佳地， 该多核苷酸编码具有 SEQ ID NO: 2所示氨基酸序列的蛋白质。 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 1-723位 的序列; (b)具有 SEQ ID NO: 1中 1-979位的序列。  In a second aspect of the present invention, there is provided a polynucleotide encoding the isolated proteins, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Homology: (a) a polynucleotide encoding the earthworm plasmin Z protein; and (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a protein having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1 to 723 in SEQ ID NO: 1; (b) a sequence having positions 1 to 979 in SEQ ID NO: 1 the sequence of.

在本发明的第三方面， 提供了含有上述多核苷酸的载体， 以及被该载体转化 或转导的宿主细胞或者被上述多核苷酸直接转化或转导的宿主细胞。  In a third aspect of the present invention, there are provided a vector containing the above-mentioned polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above-mentioned polynucleotide.

在本发明的第四方面， 提供了制备具有蚯蚓纤溶酶 z蛋白质活性的蛋白质的 方法， 该方法包含： （a)在适合表达蚯蚓纤溶酶 Z 的条件下， 培养上述被转化或转 导的宿主细胞； （b)从培养物中分离出具有蚯蚓纤溶酶 Z蛋白质活性的蛋白质。  In a fourth aspect of the present invention, there is provided a method for preparing a protein having earthworm fibrinolytic enzyme z protein activity, the method comprising: (a) culturing the above-mentioned transformed or transduced under conditions suitable for expressing earthworm fibrinolytic enzyme Z Host cells; (b) isolating a protein with earthworm fibrinolytic Z protein activity from the culture.

在本发明的第五方面， 提供了与上述的蚯蚓纤溶酶 Z蛋白质特异性结合的抗 体。  In a fifth aspect of the present invention, an antibody that specifically binds to the earthworm fibrinolytic enzyme Z protein is provided.

在本发明的第六方面， 提供了一种药物组合物， 它含有安全有效量的本发明 的蚯蚓纤溶酶 Z 以及药学上可接受的载体。 这些药物组合物可治疗血栓栓塞性疾 病等病症。  In a sixth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the earthworm plasmin Z of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat conditions such as thromboembolic diseases.

本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。 附图说明  Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. BRIEF DESCRIPTION OF THE DRAWINGS

下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所界 定的本发明范围。  The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

图 1显示了蚯蚓纤溶酶 Z的 PCR扩增条带， 泳道 1为 DNA标准；泳道 2为 PCR 扩增产物。  Figure 1 shows the PCR amplification bands of earthworm fibrinolytic enzyme Z. Lane 1 is the DNA standard; lane 2 is the PCR amplification product.

图 2显示了蚯蚓纤溶酶 Z的基因序列及其编码的蛋白质序列。  Figure 2 shows the gene sequence of earthworm fibrinolytic enzyme Z and its encoded protein sequence.

图 3显示了表达蚯蚓纤溶酶 Z的原核表达载体 pET- 28a (+)中的构建过程。 图 4显示了蚯蚓纤溶酶 Z表达产物的 SDS- PAGE图谱和 western图谱。 其中 各泳道分别是： 1.蛋白质标准分子量， 2. IPTG诱导前菌体总蛋白质， 3. IPTG 诱导后菌体总蛋白质， 4. Western图谱。 具体实施方式  Figure 3 shows the construction process of the prokaryotic expression vector pET-28a (+) expressing earthworm fibrinolytic enzyme Z. Figure 4 shows the SDS-PAGE and western maps of the earthworm fibrinolytic Z expression products. The lanes are: 1. Standard molecular weight of protein, 2. Total protein of bacteria before IPTG induction, 3. Total protein of bacteria after IPTG induction, 4. Western map. detailed description

本发明人首先从多种蚯蚓中分离了一种分子量为 25125. 0道尔顿的蚯蚓纤溶 酶（质谱测定结果）， 命名它为蚯蚓纤溶酶 Z 。 并且， 以威廉腔环蚓

_guillelini) ^ ， 测定了蚯蚓纤溶酶 Z的 N端一段氨基酸序列， 并以此为根据 合成了相应的引物，然后从蚯蚓中分离有关的 mRNA为模板，经反转录获得 cDNA， 用 PCR法扩增和克隆了蚯蚓纤溶酶 Z的基因， 并测定了基因的全序列。 然后将蚯 蚓纤溶酶 Z的编码序列***合适载体并转入合适的宿主细胞， 表达并分离了蚯蚓 纤溶酶 Z。 并通过 Western印迹分析加以鉴定。 在本发明中， 术语 "蚯蚓纤溶酶 Z " 、 "蚯蚓纤溶酶 Z蛋白质" 或 "蚯蚓纤 溶酶 Z多肽"可互换使用，都指基本上具有蚯蚓纤溶酶 Z氨基酸序列 (SEQ ID NO:2) 的多肽或蛋白质。 它们包括含有或不含起始甲硫氨酸的纤溶酶蚯蚓纤溶酶 Z 。 这 些术语还包括含有或不含有信号肽的蚯蚓纤溶酶 Z。 The inventors first isolated an earthworm fibrinolysis with a molecular weight of 25125. 0 Daltons from a variety of earthworms. Enzyme (mass spectrometry results), named it earthworm fibrinolytic enzyme Z. And, with William ’s cavity

_g uillelini) ^, the amino acid sequence at the N-terminus of earthworm fibrinolytic enzyme Z was determined, and the corresponding primers were synthesized based on this. The relevant mRNA was isolated from the earthworm as a template, and cDNA was obtained by reverse transcription. The gene of earthworm fibrinolytic enzyme Z was amplified and cloned, and the full sequence of the gene was determined. The coding sequence of earthworm fibrinolytic enzyme Z was then inserted into a suitable vector and transferred into a suitable host cell, and earthworm fibrinolytic enzyme Z was expressed and isolated. And identified by Western blot analysis. In the present invention, the terms "earthworm plasmin Z", "earthworm plasmin Z protein", or "earthworm plasmin Z polypeptide" are used interchangeably, and all refer to having an earthworm plasmin Z amino acid sequence (SEQ ID NO: 2). They include plasmin earthworm plasmin Z with or without the starting methionine. These terms also include earthworm plasmin Z with or without a signal peptide.

如本文所用， "分离的" 是指物质从其原始环境中分离出来 (如果是天然的物 质， 原始环境即是天然环境)。 如活体细胞内的天然状态下的多聚核苷酸和蛋白质 是没有分离纯化的，但同样的多聚核苷酸或蛋白质如从天然状态中同存在的其他物 质中分开， 则为分离纯化的。  As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotide and protein in the natural state in a living cell are not separated and purified, but the same polynucleotide or protein is separated and purified if separated from other substances existing in the natural state. .

如本文所用， "分离的蚯蚓纤溶酶 Z多肽或蛋白质" 是指蚯蚓纤溶酶 Z蛋白 质基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人 员能用标准的蛋白质纯化技术 (尤其是 FPLC)分离纯化出蚯蚓纤溶酶∑。  As used herein, "isolated earthworm plasmin Z polypeptide or protein" means that earthworm plasmin Z protein is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can isolate and purify the earthworm fibrinolytic enzyme Σ using standard protein purification techniques, especially FPLC.

本发明的蛋白质可以是重组蛋白质、 天然蛋白质、 合成蛋白质， 优选重组蛋 白质。 本发明的蛋白质可以是天然纯化的产物， 或是化学合成的产物， 或使用重 组技术从原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞） 中产生。 根据重组生产方案所用的宿主， 本发明的蛋白质可以是糖基化的， 或可 以是非糖基化的。 本发明的蛋白质还可包括或不包括起始的甲硫氨酸残基。  The protein of the present invention may be a recombinant protein, a natural protein, or a synthetic protein, and preferably a recombinant protein. The protein of the present invention may be a naturally purified product or a chemically synthesized product or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the protein of the invention may be glycosylated, or it may be non-glycosylated. The proteins of the invention may also or may not include the initial methionine residue.

本发明还包括蚯蚓纤溶酶 Z的片段、衍生物和类似物。 如本文所用， 术语"片 段" 、 "衍生物"和 "类似物"是指基本上保持本发明的天然蚯蚓纤溶酶 Z 相同 的生物学功能或活性的蛋白质。 本发明的蛋白质片段、 衍生物或类似物可以是 ω 有一个或多个保守或非保守性氨基酸残基 (优选保守性氨基酸残基)被取代的蛋白 质， 而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的， 或 (ii)在一 个或多个氨基酸残基中具有取代基团的蛋白质， 或 (iii)成熟蛋白质与另一个化合物 (比如延长蛋白质半衰期的化合物， 例如聚乙二醇)融合所形成的蛋白质， 或 (iv)附 加的氨基酸序列融合到此蛋白质序列而形成的蛋白质 (如前导序列或分泌序列或用 来纯化此蛋白质的序列或醸原序列， 或与抗原 IgG片段的形成的融合蛋白）。 根据 本文的教导， 这些片段、 衍生物和类似物属于本领域熟练技术人员公知的范围。 在本发明中，术语"蚯蚓纤溶酶 Z蛋白质 "指具有蚯蚓纤溶酶 Z活性的 SEQ ID NO. 2序列的蛋白质。该术语还包括具有与蚯蚓纤溶酶 Z相同功能的、 SEQ ID NO. 2序列的变异形式。 这些变异形式包括 (但并不限于)： 若干个 (通常为 1-50个， 较 佳地 1-30个， 更佳地 1-20个， 最佳地 1-10个)氨基酸的缺失、 ***和 /或取代， 以 及在 C末端和 /或 N末端添加一个或数个 (通常为 20个以内， 较佳地为 10个以内， 更佳地为 5个以内)氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基酸进行 取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加一个或 数个氨基酸通常也不会改变蛋白质的功能。 该术语还包括蚯蚓纤溶酶 Z 的活性片 段和活性衍生物。 The invention also includes fragments, derivatives and analogs of earthworm fibrinolytic enzyme Z. As used herein, the terms "fragment", "derivative" and "analog" refer to a protein that substantially retains the same biological function or activity of the natural earthworm fibrinolytic enzyme Z of the present invention. The protein fragment, derivative or analog of the present invention may be a protein in which ω has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may be It may not be encoded by the genetic code, or (ii) a protein with a substituent group in one or more amino acid residues, or (iii) a mature protein with another compound (such as a compound that extends the half-life of a protein, such as polyethylene Diol), a protein formed by fusion, or (iv) a protein formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this protein or a prion sequence, or an antigen IgG Fragment of the fusion protein). According to As taught herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art. In the present invention, the term "earthworm plasmin Z protein" refers to a protein of SEQ ID NO. 2 sequence having earthworm plasmin Z activity. The term also includes variants of the sequence of SEQ ID NO. 2 having the same function as earthworm fibrinolytic enzyme Z. These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions And / or substitutions, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, the substitution of amino acids with similar or similar properties usually does not change the function of the protein. As another example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of earthworm fibrinolytic enzyme Z.

该蛋白质的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突 变体、诱导突变体、在高或低的严紧度条件下能与蚯蚓纤溶酶 Z DNA 杂交的 DNA 所编码的蛋白、 以及利用抗蚯蚓纤溶酶 Z 蛋白质的抗血清获得的多肽或蛋白质。 本发明还提供了其他蛋白质， 如包含蚯蚓纤溶酶 Z 蛋白质或其片段的融合蛋白。 除了几乎全长的蛋白质外， 本发明还包括了蚯蚓纤溶酶 Z 蛋白质序列的可溶性片 段。 通常， 该片段具有蚯蚓纤溶酶 Z蛋白质序列的至少约 10个连续氨基酸， 通常 至少约 30个连续氨基酸， 较佳地至少约 50个连续氨基酸， 更佳地至少约 80个连 续氨基酸， 最佳地至少约 100个连续氨基酸。  Variations of this protein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA sites that can hybridize with earthworm plasmin Z DNA under high or low stringency conditions Encoded protein, and a polypeptide or protein obtained using an antiserum against earthworm plasmin Z protein. The present invention also provides other proteins, such as a fusion protein comprising an earthworm fibrinolytic enzyme Z protein or a fragment thereof. In addition to the almost full-length protein, the invention also includes soluble fragments of the earthworm fibrinolytic enzyme Z protein sequence. Generally, the fragment has at least about 10 consecutive amino acids, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, and more preferably at least about 80 consecutive amino acids, and most preferably at least about 80 consecutive amino acids. To at least about 100 consecutive amino acids.

发明还提供蚯蚓纤溶酶 Z或的类似物。 这些类似物与天然蚯蚓纤溶酶 Z蛋白 质的差别可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而有之。这些蛋白质包括天然或诱导的遗传变异体。诱导变异体可以通过各 种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或 其他已知分子生物学的技术。类似物还包括具有不同于天然 L-氨基酸的残基 (如 D- 氨基酸)的类似物， 以及具有非天然存在的或合成的氨基酸 (如 β、 y -氨基酸)的类 似物。 应理解， 本发明的蛋白质并不限于上述例举的代表性的蛋白质。  The invention also provides earthworm plasmin Z or an analogue. The differences between these analogs and the natural earthworm fibrinolytic enzyme Z protein may be differences in amino acid sequences, differences in modified forms that do not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, y-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.

修饰 (通常不改变一级结构)形式包括：体内或体外的蛋白质的化学衍生形式如 乙酰化或羧基化。修饰还包括糖基化， 如那些在蛋白质的合成和加工中或进一步加 工步骤中进行糖基化修饰而产生的蛋白质。这种修饰可以通过将蛋白质暴露于进行 糖基化的酶 (如哺乳动物的糖基化酶或去糖基化酶)而完成。 修饰形式还包括具有磷 酸化氨基酸残基 (如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修饰 从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白质。  Modified (usually unchanged primary structure) forms include chemically derived forms of proteins in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of proteins or in further processing steps. This modification can be accomplished by exposing the protein to an enzyme that undergoes glycosylation, such as mammalian glycosylation or deglycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize their solubility.

在本发明中， "蚯蚓纤溶酶 Z保守性变异蛋白质" 指与 SEQ ID NO : 2的氨基酸 序列相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸 被性质相似或相近的氨基酸所替换而形成蛋白质。这些保守性变异蛋白质最好根据 表 1进行氨基酸替换而产生。 表 1In the present invention, the "earthworm plasmin Z conservative variant protein" refers to the amino acid of SEQ ID NO: 2 Compared with sequences, there are at most 10, preferably at most 8 and more preferably at most 5 and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a protein. These conservatively mutated proteins are preferably produced by amino acid substitution according to Table 1. Table 1

本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、基 因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编 码链或非编码链。 编码成熟蛋白质的编码区序列可以与 SEQ ID ΝΟ: 1所示的编码 区序列相同或者是简并的变异体。 如本文所用， "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， 但与 SEQ ID ΝΟ: 1所示的编码区序列有差别的 核酸序列。

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature protein may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but which differs from the coding region sequence shown in SEQ ID NO: 1.

编码 SEQ ID NO:2的成熟蛋白质的多核苷酸包括： 只编码成熟蛋白质的编码 序列； 成熟蛋白质的编码序列和各种附加编码序列； 成熟蛋白质的编码序列 (和任 选的附加编码序列)以及非编码序列。 术语 "编码蛋白质的多核苷酸" 可以是包括编码此蛋白质的多核苷酸， 也可 以是还包括附加编码和 /或非编码序列的多核苷酸。 The polynucleotide encoding the mature protein of SEQ ID NO: 2 includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences); and Non-coding sequence. The term "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or a polynucleotide that also includes additional coding and / or non-coding sequences.

本发明还涉及上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 的多肽或蛋白质的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的 等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异 体和***变异体。如本领域所知的， 等位变异体是一个多核苷酸的替换形式， 它可 能是一个或多个核苷酸的取代、缺失或***，但不会从实质上改变其编码的蛋白质 的功能。  The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or proteins having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides without substantially altering the function of the protein it encodes .

本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少 70%, 更佳地至少 80%同源性的多核苷酸。本发明特别涉及在严格条件下与本发明 所述多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件"是指 :（1)在较低离子 强度和较高温度下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS, 60 °C _; 或 (2)杂交时加 有变性剂， 如 50%(v/v)甲酰胺， 0.1%小牛血清 /0.1% Ficoll， 42 °C等；或 (3)仅在两 条序列之间的同源性至少在 90%以上，更好是 95%以上时才发生杂交。 并且， 可杂 交的多核苷酸编码的蛋白质与 SEQ ID NO:2所示的成熟蛋白质有相同的生物学功 能和活性。 The present invention also relates to a polynucleotide that hybridizes to the aforementioned sequence and has at least 50%, preferably at least 70%, and more preferably at least 80% homology between the two sequences. The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C _; or (2) during hybridization Added denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the homology between only two sequences is at least 90% Above, and preferably above 95%, hybridization occurs. In addition, the protein encoded by the hybridizable polynucleotide has the same biological function and activity as the mature protein shown in SEQ ID NO: 2.

本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段" 的 长度至少含 15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个核苷酸以上。 核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 / 或分离编码蚯蚓纤溶酶 Z的多聚核苷酸。  The invention also relates to a nucleic acid fragment that hybridizes to the sequence described above. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding earthworm plasmin Z.

本发明中的蛋白质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 protein and polynucleotide in the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

本发明的蚯蚓纤溶酶 Z核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组 法或人工合成的方法获得。对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序 歹 I」， 尤其是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员 已知的常规方法所制备的 cDNA库作为模板， 扩增而得到有关序列。 也可直接通过 RT-PCR的方法扩增得到有关序列。 当序列较长时， 常常需要进行两次或多次 PCR 扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。  The full-length Z-nucleotide sequence of the earthworm fibrinolytic enzyme of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method. For the PCR amplification method, primers can be designed according to the disclosed nucleotide sequence 」I, especially the open reading frame sequence, and a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The prepared cDNA library was used as a template and amplified to obtain the relevant sequences. Relevant sequences can also be obtained directly by RT-PCR. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.

一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常 是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得 到有关序列。  Once the relevant sequences are obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通 常， 通过先合成多个小片段， 然后再进行连接可获得序列很长的片段。 目前， 已经可以完全通过化学合成来得到编码本发明蛋白质 (或其片段， 或其 衍生物)的 DNA序列。然后可将该 DNA序列引入本领域中已知的各种现有的 DNA 分子 (或如载体)和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白质序列 中。 In addition, relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short. Pass Often, long fragments can be obtained by first synthesizing multiple small fragments and then performing ligation. At present, a DNA sequence encoding a protein (or a fragment thereof, or a derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

应用 PCR技术扩增 DNA/RNA的方法 (Saiki, et al. Science 1985;230:1350-1354) 被优选用于获得本发明的基因。 特别是很难从文库中得到全长的 cDNA 时， 可优 选使用 RACE法 (RACE-cDNA末端快速扩增法)，用于 PCR的引物可根据本文所公 开的本发明的序列信息适当地选择， 并可用常规方法合成。可用常规方法如通过凝 胶电泳分离和纯化扩增的 DNA/RNA片段。  A method for amplifying DNA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) may be preferably used. The primers used for PCR may be appropriately selected based on the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

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蚯蚓纤 溶酶 Z 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蛋 白质的方法。  The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or earthworm plasmin Z coding sequence, and a method for producing the protein of the present invention by recombinant technology.

通过常规的重组 DNA技术 (Science , 1984； 224： 1431)， 可利用本发明的 多聚核苷酸序列可用来表达或生产重组的蚯蚓纤溶酶 Z 蛋白质。 一般来说有以下 步骤：  By conventional recombinant DNA technology (Science, 1984; 224: 1431), the polynucleotide sequence of the present invention can be used to express or produce recombinant earthworm plasmin Z protein. Generally there are the following steps:

(1) .用本发明的编码蚯蚓纤溶酶 z蛋白质的多核苷酸 (或变异体)， 或用含有该 多核苷酸的重组表达载体转化或转导合适的宿主细胞；  (1) using the polynucleotide (or variant) encoding the earthworm fibrinolytic enzyme z protein of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) .在合适的培养基中培养的宿主细胞；  (2) host cells cultured in a suitable medium;

(3).从培养基或细胞中分离、 纯化蛋白质。  (3). Isolate and purify protein from culture medium or cells.

本发明中， 蚯蚓纤溶酶 Z多核苷酸序列可***到重组表达载体中。 术语 "重 组表达载体"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、逆转录病毒或其他载体。在本发明中适用的载体包括但不 限于： 在细菌中表达的基于 T7 的表达载体； 在哺乳动物细胞中表达的 pMSXND 表达载体和在昆虫细胞中表达的来源于杆状病毒的载体。总之， 只要能在宿主体内 复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制 起点、 启动子、 标记基因和翻译控制元件。  In the present invention, the earthworm plasmin Z polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors expressed in bacteria; pMSXND expression vectors expressed in mammalian cells; and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.

本领域的技术人员熟知的方法能用于构建含蚯蚓纤溶酶 Z编码 DNA序列和合 适的转录 /翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合 成技术、 体内重组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动 子上， 以指导 mRNA合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启 动子； λ噬菌体 P_L启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激 酵启动子、 早期和晚期 SV40启动子、 反转录病毒的 LTRs和其他一些已知的可控 制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的 核糖体结合位点和转录终止子。 Methods known to those skilled in the art can be used to construct expression vectors containing earthworm fibrinolytic enzyme Z-encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the lambda phage _PL promoter; eukaryotic promoters including the CMV immediate early promoter, HSV thymidine Yeast promoters, early and late SV40 promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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。  In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于转 化适当的宿主细胞， 以使其能够表达蛋白质。  A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.

宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 鼠伤 寒沙门氏菌的细菌细胞； 真菌细胞如酵母； 植物细胞； 果蝇 S2或 S 9的昆虫细胞； CHO、 COS , 293细胞、 或 Bowes黑素瘤细胞的动物细胞等。  The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or S 9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells and so on.

本发明的多核苷酸在高等真核细胞中表达时， 如果在载体中***增强子序列 时将会使转录得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300 个碱基对， 作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一 侧的 100到 270个碱基对的 SV40增强子、在复制起始点晚期一侧的多瘤增强子以 及腺病毒增强子等。  When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, transcription will be enhanced. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.

本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子和宿主细 胞。  Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主 为原核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 ( &(：1₂法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl₂。 如果需 要， 转化也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染 方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be harvested after exponential growth phase, with (& (: Treatment 1 _2, steps well known in the art using another method is to use MgCl. _2. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.

获得的转化子可以用常规方法培养， 表达本发明的基因所编码的蛋白质。 根 据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞 生长的条件下进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 (如 温度转换或化学诱导)诱导选择的启动子， 将细胞再培养一段时间。  The obtained transformants can be cultured by a conventional method to express a protein encoded by the gene of the present invention. Depending on the host cells used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

在上面的方法中的重组蛋白质可在细胞内、 或在细胞膜上表达、 或分泌到细 胞外。 如果需要， 可利用其物理的、化学的和其它特性通过各种分离方法分离和纯 化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不 限于： 常规的复性处理、用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、超处理、 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。 The recombinant protein in the above method may be expressed intracellularly, or on a cell membrane, or secreted extracellularly. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, Ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

重组的蚯蚓纤溶酶 Z或蛋白质有多方面的用途。这些用途包括 (但不限于)： 直 接做为药物治疗血栓栓塞性疾病的疾病， 和用于筛选促进或对抗蚯蚓纤溶酶 Z 功 能的抗体、 蛋白质或其它配体。 用表达的重组蚯蚓纤溶酶 Z 筛选蛋白质库可用于 寻找有治疗价值的能抑制或刺激蚯蚓纤溶酶 Z功能的蛋白质分子。  Recombinant earthworm plasmin Z or protein has many uses. These uses include (but are not limited to): direct use as a drug to treat thromboembolic diseases, and to screen antibodies, proteins, or other ligands that promote or combat earthworm fibrinolytic enzyme Z function. Screening the protein library with the expressed recombinant earthworm fibrinolytic enzyme Z can be used to find therapeutic protein molecules that can inhibit or stimulate the function of earthworm fibrinolytic enzyme Z.

另一方面， 本发明还包括对蚯蚓纤溶酶 Z DNA或是其片段编码的蛋白质具有 特异性的多克隆抗体和单克隆抗体， 尤其是单克隆抗体。 这里， "特异性"是指抗 体能结合于蚯蚓纤溶酶 Z基因产物或片段。 较佳地， 指那些能与蚯蚓纤溶酶 Z基 因产物或片段结合但不识别和结合于其它非相关抗原分子的抗体。本发明中抗体包 括那些能够结合并抑制蚯蚓纤溶酶 z 的分子， 也包括那些并不影响蚯蚓纤溶酶 Z 功能的抗体。 本发明还包括那些能与修饰或未经修饰形式的蚯蚓纤溶酶 Z基因产 物结合的抗体。  On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, having specificity for the protein encoded by earthworm fibrinolytic enzyme Z DNA or fragments thereof. Here, "specificity" means that the antibody is capable of binding to the earthworm fibrinolytic enzyme Z gene product or fragment. Preferably, it refers to those antibodies that can bind to earthworm plasmin Z gene products or fragments but do not recognize and bind to other unrelated antigen molecules. The antibodies in the present invention include those molecules capable of binding and inhibiting earthworm fibrinolytic enzyme z, as well as those which do not affect the function of earthworm fibrinolytic enzyme z. The invention also includes those antibodies that are capable of binding to a modified or unmodified form of the earthworm plasmin Z gene product.

本发明不仅包括完整的单克隆或多克隆抗体， 而且还包括具有免疫活性的抗 体片段， 如 Fab'或 (Fab)₂片段； 抗体重链； 抗体轻链； 遗传工程改造的单链 Fv分 子； 或嵌合抗体， 如具有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。 The invention includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab 'or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; Or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain the antibody portion from humans.

本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。 例如， 纯化的蚯蚓纤溶酶 Z基因产物或者其具有抗原性的片段， 可被施用于动物以诱导 多克隆抗体的产生。 与之相似的， 表达蚯蚓纤溶酶 Z 或其具有抗原性的片段的细 胞可用来免疫动物来生产抗体。本发明的抗体也可以是单克隆抗体。此类单克隆抗 体可以利用杂交瘤技术来制备 。 本发明的各类抗体可以利用蚯蚓纤溶酶 z基因 产物的片段或功能区，通过常规免疫技术获得。这些片段或功能区可以利用重组方 法制备或利用蛋白质合成仪合成。 与蚯蚓纤溶酶 z基因产物的未修饰形式结合的 抗体可以用原核细胞 (例如 Co//)中生产的基因产物来免疫动物而产生； 与翻译后 修饰形式结合的抗体 (如糖基化或磷酸化的蛋白或多肽)，可以用真核细胞 (例如酵母 或昆虫细胞)中产生的基因产物来免疫动物而获得。  The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, a purified earthworm plasmin Z gene product or an antigenic fragment thereof can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing earthworm plasmin Z or its antigenic fragments can be used to immunize animals to produce antibodies. The antibody of the present invention may be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology. The various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the earthworm fibrinolytic enzyme z gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a protein synthesizer. Antibodies that bind to the unmodified form of the earthworm fibrinolytic enzyme z gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (for example, Co //); Phosphorylated proteins or polypeptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.

利用本发明蚯蚓纤溶酶 Z， 通过各种常规筛选方法， 可筛选出与蚯蚓纤溶酶 Z 发生相互作用的物质， 如受体、 抑制剂、 激动剂或拮抗剂等。  Using the earthworm fibrinolytic enzyme Z of the present invention, through various conventional screening methods, substances that interact with the earthworm fibrinolytic enzyme Z, such as receptors, inhibitors, agonists or antagonists, can be screened.

本发明蚯蚓纤溶酶 Z及其抗体、 抑制剂、 激动剂、 或拮抗剂等， 当在治疗上 进行施用（给药）时， 可提供不同的效果。 通常， 可将这些物质配制于无毒的、 惰性 的和药学上可接受的水性载体介质中， 其中 pH通常约为 5- 8，较佳地 pH约为 6-8 , 尽管 pH值可随被配制物质的性质以及待治疗的病症而有所变化。 配制好的药物组 合物可以通过常规途径进行给药，其中包括 (但并不限于）：肌内、腹膜内、静脉内 、 皮下、 皮内、 或局部给药。 The earthworm fibrinolytic enzyme Z, and the antibody, inhibitor, agonist, or antagonist of the earthworm of the present invention, when administered therapeutically (administration), can provide different effects. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5 to 8, preferably pH 6 to 8, although the pH can be varied with The nature of the formulation and the condition to be treated will vary. Formulated drug group The compounds can be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.

本发明的蛋白质可直接用于疾病治疗， 例如， 用于血栓方面的治疗。 在使用 本发明蚯蚓纤溶酶 Z时， 还可同时使用其他治疗剂， 如抗肿瘤药物等。  The protein of the present invention can be directly used for the treatment of diseases, for example, for the treatment of thrombosis. When the earthworm plasmin Z of the present invention is used, other therapeutic agents, such as antitumor drugs, can also be used simultaneously.

本发明还提供了一种药物组合物， 它含有安全有效量的本发明蚯蚓纤溶酶 Z 蛋白质以及药学上可接受的载体或赋形剂。 这类载体包括 (但并不限于)： 盐水、 缓 冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 药物制剂应与给药方式相匹配。 本发 明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的 水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方 法进行制备。 药物组合物如针剂、 溶液、 片剂和胶囊宜在无菌条件下制造。 活性成 分的给药量是治疗有效量， 例如每天约 0.1微克 /千克体重-约 5毫克 /千克体重。 此 夕卜， 本发明的蛋白质还可与其他治疗剂一起使用。  The invention also provides a pharmaceutical composition containing a safe and effective amount of the earthworm fibrinolytic enzyme Z protein of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, from about 0.1 micrograms per kilogram body weight to about 5 milligrams per kilogram body weight per day. In addition, the protein of the present invention can also be used with other therapeutic agents.

使用药物组合物时， 是将安全有效量的蚯蚓纤溶酶 Z施用于哺乳动物， 其中 该安全有效量通常至少约 10微克 /天， 而且在大多数情况下不超过约 8毫克 /千克 体重， 较佳地该剂量是约 10微克 /天-约 1毫克 /千克体重。 当然， 具体剂量还应考 虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。  When the pharmaceutical composition is used, a safe and effective amount of earthworm fibrinolytic Z is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / day, and in most cases not more than about 8 mg / kg body weight, Preferably the dose is from about 10 micrograms / day to about 1 mg / kg body weight. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health, etc., which are all within the skill of a skilled physician.

一种检测检测样品中是否存在蚯蚓纤溶酶 Z的方法是利用蚯蚓纤溶酶 Z的特 异性抗体进行检测， 它包括： 将样品与蚯蚓纤溶酶 z特异性抗体接触； 观察是否 形成抗体复合物， 形成了抗体复合物就表示样品中存在蚯蚓纤溶酶 z。 本发明的优点在于： 蚯蚓中含有 10 多种的纤溶酶。 从蚯蚓中分离纯化其中 的一种成分至单一的纯度， 在工艺上有一定的难度。 本发明克隆到蚯蚓纤溶酶 z 基因， 分析测定了其基因序列， 并成功地进行了表达。 这些研究成果， 不仅为今 后开发研究基因工程产品创造了良好的条件， 也为今后开发蚯蚓纤溶酶针剂提供 了必要的化学结构资料。 此外， 本发明的蚯蚓纤溶酶 Z为治疗血栓栓塞性疾病等 疾病提供新的治疗途径， 因而具有巨大的应用前景。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通 常按照常规条件如 Sambrook等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1  A method for detecting the presence or absence of earthworm plasmin Z in a sample is to use a specific antibody for earthworm plasmin Z, which comprises: contacting the sample with earthworm plasmin z specific antibody; and observing whether an antibody complex is formed The formation of an antibody complex indicates the presence of earthworm fibrinolytic enzyme z in the sample. The advantage of the present invention is that the earthworm contains more than 10 kinds of plasmin. It is difficult to separate and purify one of the components from earthworms to a single purity. In the present invention, the earthworm fibrinolytic enzyme z gene was cloned, its gene sequence was analyzed and determined, and it was successfully expressed. These research results not only create good conditions for future research and development of genetic engineering products, but also provide necessary chemical structure information for the future development of earthworm fibrinolytic injections. In addition, the earthworm fibrinolytic enzyme Z of the present invention provides a new therapeutic approach for the treatment of diseases such as thromboembolic diseases, and thus has great application prospects. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually performed according to the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions. Example 1

从蚯蚓中分离纯化天然的蚯蚓纤溶酶 Z 在本实施例中， 采用了以下 4 种蚯蚓种类： 钜齿远蚓 Amynthas danca tala)、 赤子爱胜虫引 {Eisenia fae tide)、 壮伟远环虫引 {Amyn thas robust us)、 威廉腔环蚓

。 在本实施例及以下实施例中， 除非特别注 明， 否则指以上四种蚯蚓。 Isolation and purification of natural earthworm fibrinolytic enzyme Z from earthworms In this embodiment, the following four types of earthworms are used: Amynthas danca tala), Eisenia fae tide, Amyn thas robust us, and William's cavity ring earthworm

. In this embodiment and the following embodiments, unless specifically noted, they refer to the above four types of earthworms.

取鲜活蚯蚓若干， 经机械粉碎， 迅速冷却至 4- 10摄氏度。加入一倍体积的有机 溶剂（丙酮）， 低温萃取出脂质、 水分、 可溶性蛋白质、 盐等杂质。 10000 rpm冷冻 离心， 并收集沉淀。 沉淀中加入等体积的水， 搅拌 10分钟， 10000 rpm冷冻离心， 收集上清液。 上清液经过滤， 采用冷冻干燥法将其冻干。 取少量冻干固体进行分析， 在 FPLC图谱上出现 9-12个蛋白峰， 按照出峰时间的不同， 相互有所区别， 这些峰 为蚯蚓纤溶酶的同工酶， 其中第三个峰具有最显著的纤溶酶活性。 用蒸馏水溶解， 经离子交换柱， 亲和层析柱， 分子筛柱等多步分离纯化， 得到蚯蚓纤溶酶精制样品。 样品经 HPLC法， 或 SDS电泳分析， 呈现单一条带。 然后， 对该蚯蚓纤溶酶用质谱测 定测定分子量， 结果为 25125. 0道尔顿。 该酶被命名为蚯蚓纤溶酶 Z。 在测试的 4 种蚯蚓中都含有这种蚯蚓纤溶酶 Z，这表明蚯蚓纤溶酶 Z是一种普遍存在的纤溶酶。 实施例 2  Take a few fresh and live earthworms, crush them mechanically, and quickly cool them to 4-10 degrees Celsius. Add one volume of organic solvent (acetone), and extract lipids, water, soluble proteins, salts and other impurities at low temperature. Freeze centrifuge at 10,000 rpm and collect the pellet. Add an equal volume of water to the pellet, stir for 10 minutes, freeze centrifuge at 10,000 rpm, and collect the supernatant. The supernatant was filtered and lyophilized by freeze-drying. Taking a small amount of lyophilized solids for analysis, 9-12 protein peaks appear on the FPLC spectrum, which are different from each other according to the time of the peaks. These peaks are isozymes of earthworm fibrinolytic enzyme, and the third peak has The most significant plasmin activity. It was dissolved in distilled water and separated and purified through multiple steps such as an ion exchange column, an affinity chromatography column, and a molecular sieve column to obtain a purified sample of earthworm fibrinolytic enzyme. The samples were analyzed by HPLC or SDS electrophoresis and showed a single band. Then, the molecular weight of the earthworm fibrinolytic enzyme was measured by mass spectrometry, and the result was 25125. 0 Dalton. The enzyme is named earthworm fibrinolytic enzyme Z. The four earthworms tested contained this earthworm fibrinolytic enzyme Z, which indicates that earthworm fibrinolytic enzyme Z is a ubiquitous fibrinolytic enzyme. Example 2

蚯蚓纤溶酶 Z的纯化和 N端 10个氨基酸序列的测定  Purification of earthworm fibrinolytic enzyme Z and determination of the N-terminal 10 amino acid sequence

鲜活蚯蚓洗净后， 搅碎。 利用有机溶剂沉淀法制得蚯蚓纤溶酶粗制剂。 接着 通过 Sephadex G- 75， DEAE- Sepharose CL-6B及 Mono Q柱分离， 纯化到蚯蚓纤 溶酶 Z样品。 纯化的蚯蚓纤溶酶 Z样品在 PE491蛋白质测序仪上经过 10个循环， 测得如下 N端序列： ILGGTHARVG (SEQ ID NO : 7)。 实施例 3  After the fresh earthworms are washed, they are crushed. A crude preparation of earthworm fibrinolytic enzyme was prepared by an organic solvent precipitation method. Sephadex G-75, DEAE-Sepharose CL-6B, and Mono Q were then used to separate the earthworm plasmin Z sample. After 10 cycles of the purified earthworm fibrinolytic Z sample on a PE491 protein sequencer, the following N-terminal sequence was measured: ILGGTHARVG (SEQ ID NO: 7). Example 3

引物的设计和合成  Design and Synthesis of Primers

据实施例 2 中测定的蚯蚓纤溶酶 Z 的 N端的序列， 设计上游寡核苷酸引物 为： 5'ATGAATCCATGATC C TGGGAG (T) GG (ATC) ACG (ACT) A (GC) A3'(SEQ ID NO:  Based on the sequence of the N-terminus of earthworm fibrinolytic enzyme Z determined in Example 2, the upstream oligonucleotide primer was designed as: 5'ATGAATCCATGATC C TGGGAG (T) GG (ATC) ACG (ACT) A (GC) A3 '(SEQ ID NO:

6), 并在成熟蛋白的 N端加上了起始密码子 位点， 以利于该基因的表达 研究和操作。 由于迄今为止缺乏蚯蚓纤溶酶 C端及其基因下游的任何信息， 本发 明人用 3'RACE方法克隆该基因。 下游引物为： 5'GACCACGCGTATCGATGTCG AC3'(SEQ ID NO: 3)。 引物的合成委托上海生工生物工程公司完成。 实施例 4 6), and a start codon site is added to the N-terminus of the mature protein to facilitate the expression research and manipulation of the gene. Due to the lack of any information on the C-terminus of the earthworm fibrinolytic enzyme and its downstream genes, the present inventors cloned the gene using the 3'RACE method. The downstream primer was: 5'GACCACGCGTATCGATGTCG AC3 '(SEQ ID NO: 3). The synthesis of primers was entrusted to Shanghai Biotech Bioengineering Company. Example 4

总 RNA的提取  Isolation of total RNA

取威廉腔环蚓一条，在液氮中研磨成粉末。取 200mg粉末，加入 4raL溶液 D (含 4mol/L异硫氰酸胍， 25m mol/L柠檬酸钠， pH 7. 0 ； 5%十二烷基肌氨酸钠， 0. l mol/Lp -巯基乙醇）， 充分匀浆后， 将溶液转移至离心管中， 4 Ό， 12, 000g 离心 5分钟。 上清吸入新的离心管中， 加入 0. 4mL 2 mol/L NaAc, Ph 4. 0, 4mL 水饱和酚和 2mL氯仿： 异戊醇（49 : 1 )混合物， 充分振荡后，冰浴 15min 。 4 °C， 10, 000g 离心 20min 。 取上层水相与等体积异丙醇混匀， -20 °C放置 lh 。 4 °C， 10， 000g离心 20min收集沉淀.沉淀重溶于 2mL溶液 D中， 65 温育 2- 3rain， 使其充分溶解， 加入 0. 2mL 2 mol/L NaAc， pH4. 0和 2mL异丙醇， 混匀后 -20 °C 放置 lh 。 4 °〇， 10, 00(^离心2(½ ， 沉淀用 75%乙醇洗涤后， 敞口于空气中放 置数分钟， 使乙醇充分挥发干净。最后溶于 DEPC (焦碳酸二乙酯）处理过的灭菌水 30μί中。取 Ιμί测 RNA的 0D₂₆。/0D₂₈。， Ι μί走甲醛变性电泳，以确定 RNA的纯度和完 整性。 实施例 5 Take a William cavity ringworm and grind it into powder in liquid nitrogen. Take 200mg powder and add 4raL solution D (containing 4mol / L guanidine isothiocyanate, 25m mol / L sodium citrate, pH 7.0; 5% sodium dodecylsarcosine, 0.1 mol / Lp-mercaptoethanol), after homogenizing, The solution was transferred to a centrifuge tube and centrifuged for 4 minutes at 12,000g for 5 minutes. The supernatant was aspirated into a new centrifuge tube, and 0.4 mL of 2 mol / L NaAc, Ph 4.0, 4 mL of water-saturated phenol and 2 mL of chloroform: isoamyl alcohol (49: 1) were added. After shaking, the solution was ice-baked for 15 min. Centrifuge at 10,000g at 4 ° C for 20min. Take the upper aqueous phase and mix with an equal volume of isopropanol, and leave it at -20 ° C for 1 h. 4 ° C, 10,000 g centrifugation for 20 minutes to collect the precipitate. The precipitate was re-dissolved in 2 mL of solution D, and incubated for 2 to 3 rain, to make it fully dissolved, 0.2 mL 2 mol / L NaAc, pH 4.0 and 2 mL isopropyl Alcohol, mix for 1 h at -20 ° C. 4 ° 〇, 10, 00 (centrifugation 2 (½), the precipitate was washed with 75% ethanol, and left open in the air for several minutes to fully evaporate the ethanol. Finally dissolved in DEPC (diethyl pyrocarbonate) treatment Take 30 μί of sterile water. Take 1 μί to measure the RNA's 0D ₂₆ / 0D _28. , Ι μί take formaldehyde denaturing electrophoresis to determine the purity and integrity of the RNA. Example 5

RT-PCR扩增蚯蚓纤溶酶 Ζ基因  RT-PCR amplification of earthworm fibrinolytic enzyme Z gene

以实施例 4中制得的威廉腔环蚓总 RNA为模板， 用宝林曼公司 3'RACE KIT反 转录， 操作按说明书进行。  The total RNA of William's columbaria vermicularis prepared in Example 4 was used as a template, and 3'RACE KIT was used for reverse transcription by Bowringman Company. The operation was performed according to the instructions.

使用实施例 3中合成的引物， 从上述所得的 cDNA为模板， 进行 PCR扩增。 总反应体系为 50μί， 其中 MgCl₂的终浓度为 1. 5m mol/L, dNTP 的终浓度为 20(^mol/L。 引物浓度为 30n mol/L， Tag酶 2单位（购自 Gibco BRL公司）。 反应 条件为： 先 94 °C变性 3min,。然后进入循环，。 94 变性 lmin ; 50 °C退火 rain ; 72 °C延伸 1. 5min， 进行 30个循环， 最后在 72 °C保温 l Omin 。 PCR产物取 5μί 进行 1%的琼脂糖电泳检查， 扩增出一条约 1000bp的 DNA片段（见图 1)。 产物以 氯仿抽提，乙醇沉淀回收，溶于 ΙΟμΙ^的灭菌水中。 实施例 6 The primers synthesized in Example 3 were used for PCR amplification from the cDNA obtained above as a template. The total reaction system was 50 μί, where the final concentration of MgCl ₂ was 1.5 m mol / L, and the final concentration of dNTP was 20 μmol / L. The concentration of the primer was 30 n mol / L, and the unit of Tagase 2 (purchased from Gibco BRL) ). The reaction conditions are: denaturation at 94 ° C for 3 min, and then entering the cycle. 94 denaturation lmin; 50 ° C annealing rain; 72 ° C extension 1.5 minutes, 30 cycles, and finally incubation at 72 ° C for 10 minutes 5 μί of the PCR product was subjected to 1% agarose electrophoresis to amplify a DNA fragment of about 1000 bp (see Figure 1). The product was extracted with chloroform, recovered by ethanol precipitation, and dissolved in 10 μl of sterilized water. Example 6

蚯蚓纤溶酶 Ζ基因的克隆  Cloning of earthworm fibrinolytic enzyme Z gene

由于扩增的是一个未知序列的核酸， 为避免基因内部出现巧合的酶切位点 导致基因被切割而破坏其完整性， 用 Τ-载体进行克隆。  Because the amplified nucleic acid is an unknown sequence, in order to avoid the occurrence of coincident restriction sites inside the gene, which may cause the gene to be cleaved and destroy its integrity, a T-vector was used for cloning.

1) Τ-载体的制备：载体 pBluescript-SK (购自 Stratagene公司） l g用 ^: oRI 切割， 等体积酚：氯仿处理， 乙醇沉淀， 溶于 50μί体系中， 内含 lOxPCR缓冲液 5μί, lOOra mol/L άΝΤΡ2μί, Taq酶 1单位。 在 70 °C保温 2h, 等体积的酚：氯仿处 理， 乙醇沉淀后，溶于 ΙΟμί灭菌水。  1) Preparation of the T-carrier: the vector pBluescript-SK (purchased from Stratagene) was cut with ^: oRI, equal volume of phenol: chloroform treatment, ethanol precipitation, dissolved in 50μί system, containing lOxPCR buffer 5μί, lOOra mol / L άΝΤΡ2μί, Taq enzyme 1 unit. Incubate at 70 ° C for 2h, treat with an equal volume of phenol: chloroform. After ethanol precipitation, dissolve in 10μl sterilized water.

2)取 PCR回收产物 2μί与自制 Τ-载体 1 连接， 转化， 经兰白斑筛选，酶 切和 PCR鉴定，获得阳性克隆。 实施例 7 2) Take the PCR recovered product 2μί and ligate it with the home-made T-vector 1, transform it, select it with blue and white spots, and enzyme Cut and PCR identification to obtain positive clones. Example 7

兔抗蚯蚓纤溶酶 1抗体的制备  Preparation of rabbit anti-earthworm plasmin-1 antibody

取体重为 2公斤左右的雄性新西兰大耳兔一只。 以 lmg蚯蚓纤溶酶 Z样品加 弗氏完全佐剂研磨成乳胶状， 在兔劲部多点注射。 饲养半个月后， 以 lmg蚯蚓纤 溶酶 Z 样品加弗氏不完全佐剂研磨成乳胶状， 再在兔劲部多点注射。 一个月后， 用 1. 5mg 蚯蚓纤溶酶 Z 样品加弗氏不完全佐剂以同样的方法加强免疫。 半个月 后，再用 lmg蚯蚓纤溶酶 Z样品加弗氏不完全佐剂再次加强免疫。饲养半个月后， 劲动脉取血， 4 °C静置过夜， 2, 000转离心 3分钟。上层血清即为兔抗蚯蚓纤溶 酶 Z抗体。 实施例 8  A male New Zealand big-eared rabbit weighing about 2 kg was taken. The 1mg earthworm plasmin Z sample was ground into latex with Freund's complete adjuvant, and injected at multiple points in the rabbit's core. After half a month's rearing, the sample was ground into latex with lmg earthworm plasmin Z sample and Freund's incomplete adjuvant, and then injected into the rabbit's core at multiple points. One month later, 1.5 mg earthworm plasmin Z sample plus Freund's incomplete adjuvant was used to boost immunity in the same way. Half a month later, another 1 mg of earthworm plasmin Z plus Freund's incomplete adjuvant was used to boost the immunity again. After half a month of breeding, blood was collected from the arterioles, left at 4 ° C overnight, and centrifuged at 2,000 rpm for 3 minutes. The upper serum is rabbit anti-earthworm plasmin Z antibody. Example 8

纤溶酶 Z基因的鉴定  Identification of plasmin Z gene

实施例 6中获得的阳性克隆委托上海基康生物技术公司测定。 基因全序列和 编码的蛋白质序列见图 2。 该基因示于 SEQ ID NO : 1， 长 924个 bp, 其中读码 框长 723bp (第 1-723位）， 编码 241个氨基酸（SEQ ID NO : 2)。 723个 bp以后为 3' -非翻译区， 有两个 polyA加尾信号 AATAAA。  The positive clones obtained in Example 6 were entrusted to Shanghai Ji Kang Biotechnology Company for determination. The complete gene sequence and the encoded protein sequence are shown in Figure 2. The gene is shown in SEQ ID NO: 1, and has a length of 924 bp, with a reading frame length of 723 bp (positions 1-723), and encodes 241 amino acids (SEQ ID NO: 2). The 3′-untranslated region after 723 bp has two polyA tail signals AATAAA.

蚯蚓纤溶酶 Z cDNA编码的蛋白质序列与另一个被称为 Uwibricus bi mast us 的 cDNA编码的蛋白质序列同源性达 54%。 因此， 这是一个新的未见报道的蚯蚓纤 溶酶基因。 从这个新基因所编码的氨基酸序列， 经电脑软件 DNASTAR所计算的分 子量为 25138. 10， 这与从蚯蚓中所提取的纤溶酶 Z的分子量大小是吻合的。 实施例 9  The earthworm plasmin Z cDNA encodes a protein sequence that is 54% homologous to a protein encoded by another cDNA called Uwibricus bi mast us. Therefore, this is a new unreported earthworm plasmin gene. From the amino acid sequence encoded by this new gene, the molecular weight calculated by computer software DNASTAR is 25138. 10, which is consistent with the molecular weight of plasmin Z extracted from earthworms. Example 9

蚯蚓纤溶酶 Z的表达和鉴定  Expression and identification of earthworm fibrinolytic enzyme Z

在该实施例中， 为进一步证实该基因的确编码了蚯蚓纤溶酶 Z, 将该基因的 翻译区在原核表达载体 pET- 28a (+) (Novagen公司）中表达， 用实施例 7中制得的 兔抗蚯蚓纤溶酶 τ抗体与表达产物进行 Western杂交鉴定。 其中质粒构建图谱见 图 3, 表达的结果见图 4 。  In this example, in order to further confirm that the gene does indeed encode earthworm fibrinolytic enzyme Z, the translation region of the gene was expressed in the prokaryotic expression vector pET-28a (+) (Novagen), and was prepared in Example 7 Western blot analysis of rabbit anti-earthworm fibrinolytic enzyme τ antibody and expression products. The plasmid construction map is shown in Figure 3, and the expression results are shown in Figure 4.

(1)表达  (1) Expression

根据全基因序列另合成两条引物， 以便于在 pET- 28a (+)中表达：上游引物 为 5'GCGAATTCATCCTGGGAGGGACGAAA3' (引物 3) (SEQ ID NO : 4) , 下游引物 为： 5'CACTCGAGTTAGTGCAGTCGGCTCCA3' (引物 4) (SEQ ID NO : 5)。 以全长 cDNA为 模板， 如上所述进行 PCR扩增. 扩增产物 fcoRlAttoI酶切后克隆于 pET-28a (+) 的 ₀RlA½oI 位点中， 酶切的阳性克隆经测序确保无误之后， 质粒转化于菌株 BL21 (DE3)中. 待细菌的 0D₆。。到 0. 6后加入 IPTG至 lm mol/L诱导基因的表达。 2. 5小时后离心， 收集菌体进行 SDS- PAGE， 考马斯亮蓝 R- 250染色。 Two additional primers were synthesized based on the entire gene sequence to facilitate expression in pET-28a (+): the upstream primer was 5'GCGAATTCATCCTGGGAGGGACGAAA3 '(primer 3) (SEQ ID NO: 4), and the downstream primer Is: 5'CACTCGAGTTAGTGCAGTCGGCTCCA3 '(primer 4) (SEQ ID NO: 5). The full-length cDNA was used as a template, and PCR amplification was performed as described above. The amplified product fcoRlAttoI was digested and cloned into the ₀ RlA½oI site of pET-28a (+). The positive clones digested were sequenced to ensure error-free plasmid transformation. In strain BL21 (DE3). 0D _{6 of the} bacteria to be treated. . After 0.6, IPTG was added to lm mol / L to induce gene expression. 2. After 5 hours of centrifugation, the cells were collected for SDS-PAGE and stained with Coomassie Brilliant Blue R-250.

(2) Western印迹  (2) Western blot

SDS-PAGE结束后， 取下电泳胶在冰浴的条件下 100V电转 2h至尼龙膜上。将 膜放入杂交袋， 加入 5mL封闭液（5%脱脂奶粉的磷酸盐缓冲液）， 在摆动平台上摇 动 lh.。 用封闭液以 1 : 1000稀释第一抗体（实施例 7中制得的兔抗蚯蚓纤溶酶 Z 抗体）。 打开杂交袋， 倒掉封闭液， 加入稀释好的第一抗体， 摇动 lh.。 用磷酸 盐缓冲液洗涤尼龙膜 3次， 每次 15mi_n 。 将洗涤好的膜与封闭液稀释好的羊抗 兔碱性磷酸酶（1 : 3000 稀释）杂交 lh. 。 膜用碱性磷酸酶缓冲液（100mmol/L Tris-HCl, H 9. 5, 5mmol/L MgCl₂, lOOmmol/L NaCl)洗涤后， 用 BCIP/NBT (5-溴 - 4 -氯- 3 -—吲哚磷酸 /氮蓝四唑）显色。 After SDS-PAGE is completed, remove the electrophoresis gel and transfer it to the nylon membrane at 100V for 2h under ice bath conditions. Place the membrane in a hybridization bag, add 5 mL of blocking solution (5% skim milk powder in phosphate buffer), and shake on a swinging platform for 1 h. Dilute the primary antibody (rabbit anti-earthworm plasmin Z antibody prepared in Example 7) with blocking solution at 1: 1000. Open the hybridization bag, discard the blocking solution, add the diluted primary antibody, and shake for 1 h. Nylon membrane was washed with phosphate buffer three times, each time 15mi _n. Hybridize the washed membrane with sheep anti-rabbit alkaline phosphatase (1: 3000 dilution) diluted in blocking solution for 1 h. The membrane was washed with alkaline phosphatase buffer (100mmol / L Tris-HCl, H 9. 5, 5mmol / L MgCl ₂ , 100mmol / L NaCl), and then the membrane was washed with BCIP / NBT (5-bromo-4 -chloro-3- —Indole phosphate / nitro blue tetrazole).

杂交结果显示，表达的蛋白质与蚯蚓纤溶酶 Z抗体专一性地结合（见图 4)。 实施例 10  The hybridization results showed that the expressed protein specifically bound to the earthworm fibrinolytic Z antibody (see Figure 4). Example 10

不同来源蚯蚓纤溶酶的活性测定  Determination of fibrinolytic activity of earthworms from different sources

按 Deogny 等 人 所 描 述 的 纤 维 蛋 白 平 板 法 （Clinica Chiraica Acta, 1975, 60, 85) ,根据溶圈的面积大小计算蚯蚓纤溶酶的活性。 所采用的样品 为赤子爱胜蚓纤溶酶及蚯蚓纤溶酶 Z， 活力测定的结果分别为： 赤子爱胜蚓纤溶 酶为 21， 000醒 7mg蛋白质， 蚯蚓纤溶酶为 Z 33， 000mm7mg蛋白质。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。  According to the fiber protein plate method (Clinica Chiraica Acta, 1975, 60, 85) described by Deogny et al., The activity of earthworm fibrinolytic enzyme was calculated according to the size of the lysate circle. The samples used were Eisenia foetida fibrinolytic enzyme and earthworm fibrinolytic enzyme Z, and the results of viability determination were: Eisenia foetida fibrinolytic enzyme was 21,000 and 7 mg protein, and earthworm fibrinolytic enzyme was Z 33,000 mm protein. All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

权 利 要 求 书 Claim

1.一种分离的蚯蚓纤溶酶 Z蛋白质， 其特征在于， 它包含： 具有 SEQ ID NO: 2 氨基酸序列的蛋白质、 或其保守性变异蛋白质、 或其活性片段、 或其活性衍生物。 An isolated earthworm plasmin Z protein, characterized in that it comprises: a protein having the amino acid sequence of SEQ ID NO: 2, or a conservative variant protein thereof, or an active fragment thereof, or an active derivative thereof.

2.如权利要求 1所述的蛋白质， 其特征在于， 该蛋白质是具有 SEQ ID NO: 2氨 基酸序列的蛋白质。  The protein according to claim 1, wherein the protein is a protein having the amino acid sequence of SEQ ID NO: 2.

3.—种分离的多核苷酸， 其特征在于， 它编码权利要求 1所述的蚯蚓纤溶酶 Z。  3. An isolated polynucleotide, characterized in that it encodes the earthworm fibrinolytic enzyme Z according to claim 1.

4.如权利要求 3所述的多核苷酸，其特征在于，该多核苷酸编码具有 SEQ ID NO: 2所示氨基酸序列的蛋白质。  The polynucleotide according to claim 3, wherein the polynucleotide encodes a protein having the amino acid sequence shown in SEQ ID NO: 2.

5.如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸含有 SEQ ID NO: 1 中 1-723位的序列。  The polynucleotide according to claim 3, wherein the polynucleotide comprises a sequence from positions 1 to 723 in SEQ ID NO: 1.

6.—种载体， 其特征在于， 它含有权利要求 3所述的多核苷酸。  6. A vector comprising the polynucleotide according to claim 3.

7.—种遗传工程化的宿主细胞， 其特征在于， 它含有权利要求 6所述的载体。 7. A genetically engineered host cell, characterized in that it contains the vector according to claim 6.

8. 一种具有蚯蚓纤溶酶 Z蛋白质活性的蛋白质的制备方法， 其特征在于， 该 方法包含： 8. A method for preparing a protein having earthworm fibrinolytic enzyme Z protein activity, characterized in that the method comprises:

(a)在适合表达蚯蚓纤溶酶 Z的条件下， 培养权利要求 7所述的宿主细胞； (a) culturing the host cell of claim 7 under conditions suitable for expression of earthworm plasmin Z;

(b)从培养物中分离出具有蚯蚓纤溶酶 Z蛋白质活性的蛋白质。 (b) A protein having earthworm plasmin Z protein activity is isolated from the culture.

9.一种能与权利要求 1所述的蚯蚓纤溶酶 Z特异性结合的抗体。  An antibody capable of specifically binding to earthworm plasmin Z according to claim 1.

10.—种药物组合物， 其特征在于， 它含有安全有效量的权利要求 1所述的蛋 白质以及药学上可接受的载体。  A pharmaceutical composition, characterized in that it contains a safe and effective amount of the protein according to claim 1 and a pharmaceutically acceptable carrier.