CN1407095A - Polypeptide-fructose-1,6-diphosphatase-16.61 and polynucleotide for encoding it - Google Patents

Abstract

Translated fromChinese

本发明公开了一种多肽——果糖－1，6－二磷酸酶－16.61，编码此多肽的多核苷酸和经DNA重组技术产生这种多肽的方法。本发明还公开了此多肽用于治疗多种疾病的方法，如恶性肿瘤，血液病，HIV感染和免疫性疾病和各类炎症等。本发明还公开了抗此多肽的拮抗剂及其治疗作用。本发明还公开了编码这种新的果糖－1，6－二磷酸酶－16.61的多核苷酸的用途。The invention discloses a polypeptide-fructose-1,6-diphosphatase-16.61, a polynucleotide encoding the polypeptide and a method for producing the polypeptide through DNA recombination technology. The invention also discloses a method for using the polypeptide to treat various diseases, such as malignant tumors, blood diseases, HIV infection, immune diseases and various inflammations. The invention also discloses an antagonist against the polypeptide and its therapeutic effect. The invention also discloses the application of the polynucleotide encoding the novel fructose-1,6-bisphosphatase-16.61.

Description

Translated fromChinese

一种多肽——果糖-1，6-二磷酸酶-16.61和编码这种多肽的多核苷酸A kind of polypeptide-fructose-1,6-bisphosphatase-16.61 and polynucleotide encoding this polypeptide

                             技术领域Technical field

本发明属于生物技术领域，具体地说，本发明描述了一种新的多肽——果糖-1，6-二磷酸酶-16.61，以及编码此多肽的多核苷酸序列。本发明还涉及此多核苷酸和多肽的制备方法和应用。The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-fructose-1,6-bisphosphatase-16.61, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the preparation method and application of the polynucleotide and polypeptide.

                             背景技术 Background technique

果糖-1，6-二磷酸酶-16.61(FBPase)(EC 3.1.3.11)是糖原异生的调控酶类，催化果糖-1，6-二磷酸水解产生果糖-6-磷酸。该过程存在于大多数生物的各种代谢途径。(Benkovic S.J.，DeMaine M.M.Adv.Enzymol.53：45-82(1982).)景天庚酮糖-1，7-二磷酸酶(SBPase)(EC 3.1.3.37)存在于植物叶绿体和光能自养细菌中，在卡尔文戊糖磷酸还原循环反应中催化景天庚酮糖-1，7-二磷酸水解为景天庚酮糖-7-磷酸，该酶的结构和功能均与果糖-1，6-二磷酸酶-16.61相关。(Raines C.A.，Lloyd J.C.，Willingham N.M.，Potts S.，Dyer T.A.Eur.J.Biochem.205：1053-1059(1992).)Fructose-1,6-bisphosphatase-16.61 (FBPase) (EC 3.1.3.11) is a regulatory enzyme of gluconeogenesis, which catalyzes the hydrolysis of fructose-1,6-bisphosphate to produce fructose-6-phosphate. This process is present in various metabolic pathways of most organisms. (Benkovic S.J., DeMaine M.M.Adv.Enzymol.53:45-82(1982).) Sedoheptulose-1,7-bisphosphatase (SBPase) (EC 3.1.3.37) exists in plant chloroplasts and photoautotrophs In bacteria, it catalyzes the hydrolysis of sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate in the Calvin pentose phosphate reduction cycle reaction. The structure and function of the enzyme are similar to those of fructose-1, 6-bisphosphatase-16.61 related. (Raines C.A., Lloyd J.C., Willingham N.M., Potts S., Dyer T.A. Eur. J. Biochem. 205:1053-1059 (1992).)

在哺乳动物的果糖-1，6-二磷酸酶-16.61中，一赖氨酸残基与该酶的催化机制密切相关，该残基周围区域的序列存在在各生物钟中高度保守，而在一些细菌中该赖氨酸活性位点被精氨酸所取代。上述特征性序列如下：[AG]-[RK]-L-x(1，2)-[LIV]-[FY]-E-x(2)-P-[LIVM]-[GSA](其中K/R使该酶活性位点残基)。该果糖-1，6-二磷酸酶-16.61的Cys128，Tyr113，Tyr244，Tyr264，Tyr215，Tyr331，Lys274，arg243，His253，His311残基的化学修饰与酶的活性有密切关联。(Raines C.A.，Lloyd J.C.，Willingham N.M.，Potts S.，Dyer T.A.Eur.J.Biochem.205：1053-1059(1992).)In mammalian fructose-1,6-bisphosphatase-16.61, a lysine residue is closely related to the catalytic mechanism of the enzyme, and the sequence of the region around this residue is highly conserved in various biological clocks, while in some In bacteria the lysine active site is replaced by arginine. The above-mentioned characteristic sequence is as follows: [AG]-[RK]-L-x(1,2)-[LIV]-[FY]-E-x(2)-P-[LIVM]-[GSA] (wherein K/R makes the enzyme active site residues). The chemical modifications of Cys128, Tyr113, Tyr244, Tyr264, Tyr215, Tyr331, Lys274, arg243, His253, His311 residues of the fructose-1,6-bisphosphatase-16.61 are closely related to the activity of the enzyme. (Raines C.A., Lloyd J.C., Willingham N.M., Potts S., Dyer T.A. Eur. J. Biochem. 205:1053-1059 (1992).)

1992年，Raines C.A.，Lloyd J.C.等研究发现果糖-1，6-二磷酸酶-16.61的活性由腺苷单磷酸(AMP)和果糖-2，6-二磷酸(Fru-2，6-P₂)调控，其中AMP是通过变构来抑制酶活性，而果糖-2，6-二磷酸的调控机制有待进一步研究。该酶在中性PH环境中最具催化活性。In 1992, Raines CA, Lloyd JC and others found that the activity of fructose-1,6-bisphosphatase-16.61 was controlled by adenosine monophosphate (AMP) and fructose-2,6-bisphosphate (Fru-2,6-P₂ ) regulation, in which AMP inhibits enzyme activity through allosterism, and the regulation mechanism of fructose-2,6-bisphosphate remains to be further studied. The enzyme is most catalytically active in a neutral pH environment.

本发明的多肽含有上述保守的特征性功能域，并且具有与果糖-1，6-二磷酸酶-16.61家族成员相似的生物学功能，因而被认为是果糖-1，6-二磷酸酶-16.61家族的新成员，催化果糖-1，6-二磷酸水解产生果糖-6-磷酸，与大多数生物的各种代谢途径密切相关，若发生变异或表达异常，则会影响生物体的一系列代谢活动，引起生理功能紊乱，导致各种疾病的发生。The polypeptide of the present invention contains the above-mentioned conserved characteristic functional domains, and has biological functions similar to members of the fructose-1,6-bisphosphatase-16.61 family, so it is considered to be a fructose-1,6-bisphosphatase-16.61 A new member of the family, catalyzes the hydrolysis of fructose-1,6-bisphosphate to produce fructose-6-phosphate, which is closely related to various metabolic pathways of most organisms. If it is mutated or expressed abnormally, it will affect a series of metabolisms of organisms Activities, causing physiological dysfunction, leading to the occurrence of various diseases.

由于如上所述果糖-1，6-二磷酸酶-16.61蛋白在机体内重要功能中起重要作用，而且相信这些调节过程中涉及大量的蛋白，因而本领域中一直需要鉴定更多参与这些过程的果糖-1，6-二磷酸酶-16.61蛋白，特别是鉴定这种蛋白的氨基酸序列。新果糖-1，6-二磷酸酶-16.61蛋白编码基因的分离也为研究确定该蛋白在健康和疾病状态下的作用提供了基础。这种蛋白可能构成开发疾病诊断和/或治疗药的基础，因此分离其编码DNA是非常重要的。Since the fructose-1,6-bisphosphatase-16.61 protein plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more proteins involved in these processes. Fructose-1,6-bisphosphatase-16.61 protein, especially the amino acid sequence of this protein was identified. The isolation of the gene encoding the new fructose-1,6-bisphosphatase-16.61 protein also provides the basis for studies to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and/or therapeutic drugs for diseases, so it is very important to isolate its coding DNA.

                            发明内容Contents of Invention

本发明的一个目的是提供分离的新的多肽——果糖-1，6-二磷酸酶-16.61以及其片段、类似物和衍生物。It is an object of the present invention to provide isolated novel polypeptides, fructose-1,6-bisphosphatase-16.61 and fragments, analogs and derivatives thereof.

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

本发明的另一个目的是提供含有编码果糖-1，6-二磷酸酶-16.61的多核苷酸的重组载体。Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding fructose-1,6-bisphosphatase-16.61.

本发明的另一个目的是提供含有编码果糖-1，6-二磷酸酶-16.61的多核苷酸的基因工程化宿主细胞。Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding fructose-1,6-bisphosphatase-16.61.

本发明的另一个目的是提供生产果糖-1，6-二磷酸酶-16.61的方法。Another object of the present invention is to provide a method for producing fructose-1,6-bisphosphatase-16.61.

本发明的另一个目的是提供针对本发明的多肽——果糖-1，6-二磷酸酶-16.61的抗体。Another object of the present invention is to provide antibodies against the polypeptide of the present invention - fructose-1,6-bisphosphatase-16.61.

本发明的另一个目的是提供了针对本发明多肽——果糖-1，6-二磷酸酶-16.61的模拟化合物、拮抗剂、激动剂、抑制剂。Another object of the present invention is to provide mimetic compounds, antagonists, agonists and inhibitors against the polypeptide of the present invention-fructose-1,6-bisphosphatase-16.61.

本发明的另一个目的是提供诊断治疗与果糖-1，6-二磷酸酶-16.61异常相关的疾病的方法。Another object of the present invention is to provide methods for the diagnosis and treatment of diseases associated with fructose-1,6-bisphosphatase-16.61 abnormalities.

本发明涉及一种分离的多肽，该多肽是人源的，它包含：具有<210>2氨基酸序列的多肽、或其保守性变体、生物活性片段或衍生物。较佳地，该多肽是具有<210>2氨基酸序列的多肽。The present invention relates to an isolated polypeptide, which is of human origin, comprising: a polypeptide with <210>2 amino acid sequence, or its conservative variant, biologically active fragment or derivative. Preferably, the polypeptide is a polypeptide having a <210>2 amino acid sequence.

本发明还涉及一种分离的多核苷酸，它包含选自下组的一种核苷酸序列或其变体：The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence or variant thereof selected from the group consisting of:

(a)编码具有<210>2氨基酸序列的多肽的多核苷酸；(a) a polynucleotide encoding a polypeptide having a <210>2 amino acid sequence;

(b)与多核苷酸(a)互补的多核苷酸；(b) a polynucleotide complementary to polynucleotide (a);

(c)与(a)或(b)的多核苷酸序列具有至少70％相同性的多核苷酸。(c) A polynucleotide having at least 70% identity to the polynucleotide sequence of (a) or (b).

更佳地，该多核苷酸的序列是选自下组的一种：(a)具有<210>1中55-510位的序列；和(b)具有<210>1中1-1231位的序列。More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having a sequence of 55-510 in <210>1; and (b) having a sequence of 1-1231 in <210>1 sequence.

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

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

本发明还涉及一种筛选的模拟、激活、拮抗或抑制果糖-1，6-二磷酸酶-16.61蛋白活性的化合物的方法，其包括利用本发明的多肽。本发明还涉及用该方法获得的化合物。The present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of fructose-1,6-bisphosphatase-16.61 protein, which includes using the polypeptide of the present invention. The invention also relates to the compounds obtained by this method.

本发明还涉及一种体外检测与果糖-1，6-二磷酸酶-16.61蛋白异常表达相关的疾病或疾病易感性的方法，包括检测生物样品中所述多肽或其编码多核苷酸序列中的突变，或者检测生物样品中本发明多肽的量或生物活性。The present invention also relates to a method for in vitro detection of diseases or disease susceptibility related to the abnormal expression of fructose-1,6-bisphosphatase-16.61 protein, comprising detecting the polypeptide or its coding polynucleotide sequence in biological samples Mutation, or detection of the amount or biological activity of the polypeptide of the present invention in a biological sample.

本发明也涉及一种药物组合物，它含有本发明多肽或其模拟物、激活剂、拮抗剂或抑制剂以及药学上可接受的载体。The present invention also relates to a pharmaceutical composition, which contains the polypeptide of the present invention or its mimetic, activator, antagonist or inhibitor and a pharmaceutically acceptable carrier.

本发明还涉及本发明的多肽和/或多核苷酸在制备用于治疗癌症、发育性疾病或免疫性疾病或其它由于果糖-1，6-二磷酸酶-16.61表达异常所引起疾病的药物的用途。The present invention also relates to the use of the polypeptide and/or polynucleotide of the present invention in the preparation of drugs for treating cancer, developmental diseases or immune diseases or other diseases caused by abnormal expression of fructose-1,6-bisphosphatase-16.61 use.

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

本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义：“核酸序列”是指寡核苷酸、核苷酸或多核苷酸及其片段或部分，也可以指基因组或合成的DNA或RNA，它们可以是单链或双链的，代表有义链或反义链。类似地，术语“氨基酸序列”是指寡肽、肽、多肽或蛋白质序列及其片段或部分。当本发明中的“氨基酸序列”涉及一种天然存在的蛋白质分子的氨基酸序列时，这种“多肽”或“蛋白质”不意味着将氨基酸序列限制为与所述蛋白质分子相关的完整的天然氨基酸。Unless otherwise specified, the following terms used in this specification and claims have the following meanings: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to genomic or synthetic DNA or RNA, which can be single- or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to oligopeptide, peptide, polypeptide or protein sequences and fragments or portions thereof. When "amino acid sequence" in the present invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with said protein molecule .

蛋白质或多核苷酸“变体”是指一种具有一个或多个氨基酸或核苷酸改变的氨基酸序列或编码它的多核苷酸序列。所述改变可包括氨基酸序列或核苷酸序列中氨基酸或核苷酸的缺失、插入或替换。变体可具有“保守性”改变，其中替换的氨基酸具有与原氨基酸相类似的结构或化学性质，如用亮氨酸替换异亮氨酸。变体也可具有非保守性改变，如用色氨酸替换甘氨酸。A protein or polynucleotide "variant" refers to an amino acid sequence or a polynucleotide sequence encoding it having one or more amino acid or nucleotide changes. The alterations may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. A variant may have "conservative" changes in which a substituted amino acid has similar structural or chemical properties to the original amino acid, eg, a leucine is replaced by an isoleucine. Variants may also have non-conservative changes, such as replacing glycine with tryptophan.

“缺失”是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺失。"Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or a nucleotide sequence.

“插入”或“添加”是指在氨基酸序列或核苷酸序列中的改变导致与天然存在的分子相比，一个或多个氨基酸或核苷酸的增加。“替换”是指由不同的氨基酸或核苷酸替换一个或多个氨基酸或核苷酸。"Insertion" or "addition" refers to a change in amino acid sequence or nucleotide sequence that results in the addition of one or more amino acids or nucleotides compared to the naturally occurring molecule. "Substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

“生物活性”是指具有天然分子的结构、调控或生物化学功能的蛋白质。类似地，术语“免疫学活性”是指天然的、重组的或合成蛋白质及其片段在合适的动物或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。"Biologically active" refers to a protein that possesses the structural, regulatory, or biochemical functions of a native molecule. Similarly, the term "immunological activity" refers to the ability of a native, recombinant or synthetic protein and fragments thereof to induce a specific immune response and bind to specific antibodies in a suitable animal or cell.

“激动剂”是指当与果糖-1，6-二磷酸酶-16.61结合时，一种可引起该蛋白质改变从而调节该蛋白质活性的分子。激动剂可以包括蛋白质、核酸、碳水化合物或任何其它可结合果糖-1，6-二磷酸酶-16.61的分子。"Agonist"refers to a molecule that, when bound to fructose-1,6-bisphosphatase-16.61, causes changes in the protein thereby modulating the activity of the protein. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that bind fructose-1,6-bisphosphatase-16.61.

“拮抗剂”或“抑制物”是指当与果糖-1，6-二磷酸酶-16.61结合时，一种可封闭或调节果糖-1，6-二磷酸酶-16.61的生物学活性或免疫学活性的分子。拮抗剂和抑制物可以包括蛋白质、核酸、碳水化合物或任何其它可结合果糖-1，6-二磷酸酶-16.61的分子。"Antagonist" or "inhibitor" means an agent that, when bound to fructose-1,6-bisphosphatase-16.61, blocks or modulates the biological activity or immunity of fructose-1,6-bisphosphatase-16.61 active molecules. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that bind fructose-1,6-bisphosphatase-16.61.

“调节”是指果糖-1，6-二磷酸酶-16.61的功能发生改变，包括蛋白质活性的升高或降低、结合特性的改变及果糖-1，6-二磷酸酶-16.61的任何其它生物学性质、功能或免疫性质的改变。"Modulation" means a change in the function of fructose-1,6-bisphosphatase-16.61, including an increase or decrease in protein activity, a change in binding properties and any other biological Changes in biological, functional or immunological properties.

＂基本上纯＂是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化果糖-1，6-二磷酸酶-16.61。基本上纯的果糖-1，6-二磷酸酶-16.61在非还原性聚丙烯酰胺凝胶上能产生单一的主带。果糖-1，6-二磷酸酶-16.61多肽的纯度可用氨基酸序列分析。"Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify fructose-1,6-bisphosphatase-16.61 using standard protein purification techniques. Substantially pure fructose-1,6-bisphosphatase-16.61 gave a single major band on non-reducing polyacrylamide gels. The purity of fructose-1,6-bisphosphatase-16.61 polypeptide can be analyzed by amino acid sequence.

“互补的”或“互补”是指在允许的盐浓度和温度条件下通过碱基配对的多核苷酸天然结合。例如，序列“C-T-G-A”可与互补的序列“G-A-C-T”结合。两个单链分子之间的互补可以是部分的或全部的。核酸链之间的互补程度对于核酸链之间杂交的效率及强度有明显影响。"Complementary" or "complementary" refers to the natural association of polynucleotides through base pairing under permissive conditions of salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or total. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

“同源性”是指互补的程度，可以是部分同源或完全同源。“部分同源”是指一种部分互补的序列，其至少可部分抑制完全互补的序列与靶核酸的杂交。这种杂交的抑制可通过在严格性程度降低的条件下进行杂交(Southern印迹或Northern印迹等)来检测。基本上同源的序列或杂交探针可竞争和抑制完全同源的序列与靶序列在的严格性程度降低的条件下的结合。这并不意味严格性程度降低的条件允许非特异性结合，因为严格性程度降低的条件要求两条序列相互的结合为特异性或选择性相互作用。"Homology" refers to the degree of complementarity, which may be partial or complete. "Partially homologous"refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization under conditions of reduced stringency (Southern blot or Northern blot, etc.). Substantially homologous sequences or hybridization probes can compete and inhibit binding of a fully homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions of reduced stringency permit non-specific binding, since conditions of reduced stringency require the binding of two sequences to each other for a specific or selective interaction.

“相同性百分率”是指在两种或多种氨基酸或核酸序列比较中序列相同或相似的百分率。可用电子方法测定相同性百分率，如通过MEGALIGN程序(Lasergene softwarepackage，DNASTAR，Inc.，Madison Wis.)。MEGALIGN程序可根据不同的方法如Cluster法比较两种或多种序列(Higgins，D.G.和P.M.Sharp(1988)Gene 73：237-244)。Cluster法通过检查所有配对之间的距离将各组序列排列成簇。然后将各簇以成对或成组分配。两个氨基酸序列如序列A和序列B之间的相同性百分率通过下式计算：

"Percent identity" refers to the percentage of sequences that are identical or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). MEGALIGN program can compare two or more sequences according to different methods such as Cluster method (Higgins, DG and PMSharp (1988) Gene 73:237-244). The Cluster method arranges sets of sequences into clusters by examining the distances between all pairs. Clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:

也可以通过Cluster法或用本领域周知的方法如Jotun Hein测定核酸序列之间的相同性百分率(Hein J.，(1990)Methods in emzumology 183：625-645)。The percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumology 183:625-645).

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

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

“衍生物”是指HFP或编码其的核酸的化学修饰物。这种化学修饰物可以是用烷基、酰基或氨基替换氢原子。核酸衍生物可编码保留天然分子的主要生物学特性的多肽。"Derivative" refers to a chemical modification of HFP or the nucleic acid encoding it. Such chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain key biological properties of the native molecule.

“抗体”是指完整的抗体分子及其片段，如Fa、F(ab’)₂及Fv，其能特异性结合果糖-1，6-二磷酸酶-16.61的抗原决定簇。"Antibody" refers to the whole antibody molecule and its fragments, such as Fa, F(ab')₂ and Fv, which can specifically bind the epitope of fructose-1,6-bisphosphatase-16.61.

“人源化抗体”是指非抗原结合区域的氨基酸序列被替换变得与人抗体更为相似，但仍保留原始结合活性的抗体。"Humanized antibody" refers to an antibody in which the amino acid sequence of the non-antigen-binding region has been replaced to become more similar to a human antibody, but still retains the original binding activity.

“分离的”一词指将物质从它原来的环境(例如，若是自然产生的就指其天然环境)之中移出。比如说，一个自然产生的多核苷酸或多肽存在于活动物中就是没有被分离出来，但同样的多核苷酸或多肽同一些或全部在自然系统中与之共存的物质分开就是分离的。这样的多核苷酸可能是某一载体的一部分，也可能这样的多核苷酸或多肽是某一组合物的一部分。既然载体或组合物不是它天然环境的成分，它们仍然是分离的。The term "isolated" refers to the removal of a substance from its original environment (eg, its natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in a natural system is isolated. Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

如本发明所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

如本文所用，“分离的果糖-1，6-二磷酸酶-16.61”是指果糖-1，6-二磷酸酶-16.61基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化果糖-1，6-二磷酸酶-16.61。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。果糖-1，6-二磷酸酶-16.61多肽的纯度能用氨基酸序列分析。As used herein, "isolated fructose-1,6-bisphosphatase-16.61" means that fructose-1,6-bisphosphatase-16.61 is substantially free of other proteins, lipids, carbohydrates or other proteins, lipids, carbohydrates or other proteins with which it is naturally associated. substance. Those skilled in the art can purify fructose-1,6-bisphosphatase-16.61 using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the fructose-1,6-bisphosphatase-16.61 polypeptide can be analyzed by amino acid sequence.

本发明提供了一种新的多肽——果糖-1，6-二磷酸酶-16.61，其基本上是由<210>2所示的氨基酸序列组成的。本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。The present invention provides a new polypeptide-fructose-1,6-bisphosphatase-16.61, which basically consists of the amino acid sequence shown in <210>2. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

本发明还包括果糖-1，6-二磷酸酶-16.61的片段、衍生物和类似物。如本发明所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的果糖-1，6-二磷酸酶-16.61相同的生物学功能或活性的多肽。本发明多肽的片段、衍生物或类似物可以是：(I)这样一种，其中一个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代，并且取代的氨基酸可以是也可以不是由遗传密码子编码的；或者(II)这样一种，其中一个或多个氨基酸残基上的某个基团被其它基团取代包含取代基；或者(III)这样一种，其中成熟多肽与另一种化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合；或者(IV)这样一种，其中附加的氨基酸序列融合进成熟多肽而形成的多肽序列(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列)通过本文的阐述，这样的片段、衍生物和类似物被认为在本领域技术人员的知识范围之内。Fragments, derivatives and analogs of fructose-1,6-bisphosphatase-16.61 are also encompassed by the present invention. As used in the present invention, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the fructose-1,6-bisphosphatase-16.61 of the present invention. Fragments, derivatives or analogs of polypeptides of the present invention may be: (I) one wherein one or more amino acid residues are substituted by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted which may or may not be encoded by the genetic code; or (II) one in which a certain group on one or more amino acid residues is replaced by another group comprising substituents; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused to the mature polypeptide ( Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the description herein such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence.

本发明提供了分离的核酸(多核苷酸)，基本由编码具有<210>2氨基酸序列的多肽的多核苷酸组成。本发明的多核苷酸序列包括<210>1的核苷酸序列。本发明的多核苷酸是从人胎脑组织的cDNA文库中发现的。它包含的多核苷酸序列全长为1231个碱基，其开放读框55-510编码了87个氨基酸。此多肽具有MOTIF的特征序列，可推断出该果糖-1，6-二磷酸酶-16.61具有MOTIF所代表的结构和功能。The present invention provides an isolated nucleic acid (polynucleotide) consisting essentially of a polynucleotide encoding a polypeptide having a <210>2 amino acid sequence. The polynucleotide sequence of the present invention includes the nucleotide sequence of <210>1. The polynucleotides of the present invention were discovered from a cDNA library of human fetal brain tissue. The full-length polynucleotide sequence it contains is 1231 bases, and its open reading frame 55-510 encodes 87 amino acids. This polypeptide has the characteristic sequence of MOTIF, and it can be deduced that the fructose-1,6-bisphosphatase-16.61 has the structure and function represented by MOTIF.

本发明的多核苷酸可以是DNA形式或是RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的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。编码成熟多肽的编码区序列可以与<210>1所示的编码区序列相同或者是简并的变异体。如本发明所用，“简并的变异体”在本发明中是指编码具有<210>2的蛋白质或多肽，但与<210>1所示的编码区序列有差别的核酸序列。A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence represented by <210>1 or a degenerate variant. As used in the present invention, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein or polypeptide having <210>2, but differs from the sequence of the coding region represented by <210>1.

编码<210>2的成熟多肽的多核苷酸包括：只有成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。The polynucleotide encoding the mature polypeptide of <210>2 includes: the coding sequence of the mature polypeptide only; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and the non-coding sequence sequence.

术语“编码多肽的多核苷酸”是指包括编码此多肽的多核苷酸和包括附加编码和/或非编码序列的多核苷酸。The term "polynucleotide encoding a polypeptide" is intended to include polynucleotides encoding the polypeptide and polynucleotides including additional coding and/or non-coding sequences.

本发明还涉及上述描述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片断、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。The present invention also relates to variants of the polynucleotides described above, which encode polypeptides having the same amino acid sequence as those of the present invention or fragments, analogs and derivatives of polypeptides. 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 which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

本发明还涉及与以上所描述的序列杂交的多核苷酸(两个序列之间具有至少50％，优选具有70％的相同性)。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加用变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在95％以上，更好是97％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与SEQID NO：2所示的成熟多肽有相同的生物学功能和活性。The invention also relates to polynucleotides which hybridize to the sequences described above (with at least 50%, preferably 70% identity between the two sequences). The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with Use a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

本发明还涉及与以上所描述的序列杂交的核酸片段。如本发明所用，”核酸片段”的长度至少含10个核苷酸，较好是至少20-30个核苷酸，更好是至少50-60个核苷酸，最好是至少100个核苷酸以上。核酸片段也可用于核酸的扩增技术(如PCR)以确定和/或分离编码果糖-1，6-二磷酸酶-16.61的多核苷酸。The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, the length of "nucleic acid fragment" contains at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides glycosides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding fructose-1,6-bisphosphatase-16.61.

本发明中的多肽和多核苷酸优选以分离的形式提供，更佳地被纯化至均质。The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

本发明的编码果糖-1，6-二磷酸酶-16.61的特异的多核苷酸序列能用多种方法获得。例如，用本领域熟知的杂交技术分离多核苷酸。这些技术包括但不局限于：1)用探针与基因组或cDNA文库杂交以检出同源的多核苷酸序列，和2)表达文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸片段。The specific polynucleotide sequence encoding fructose-1,6-bisphosphatase-16.61 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotides with common structural features acid fragments.

本发明的DNA片段序列也能用下列方法获得：1)从基因组DNA分离双链DNA序列；2)化学合成DNA序列以获得所述多肽的双链DNA。The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of the polypeptide.

上述提到的方法中，分离基因组DNA最不常用。DNA序列的直接化学合成是经常选用的方法。更经常选用的方法是cDNA序列的分离。分离感兴趣的cDNA的标准方法是从高表达该基因的供体细胞分离mRNA并进行逆转录，形成质粒或噬菌体cDNA文库。提取mRNA的方法已有多种成熟的技术，试剂盒也可从商业途径获得(Qiagene)。而构建cDNA文库也是通常的方法(Sambrook，et al.，MolecularCloning，A Laboratory Manual，Cold Spring Harbor Laboratory.New York，1989)。还可得到商业供应的cDNA文库，如Clontech公司的不同cDNA文库。当结合使用聚合酶反应技术时，即使极少的表达产物也能克隆。Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The method of choice more often is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit is also available from commercial sources (Qiagene). And constructing cDNA library is also common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as various cDNA libraries from the company Clontech. When combined with polymerase reaction technology, even minimal expression products can be cloned.

可用常规方法从这些cDNA文库中筛选本发明的基因。这些方法包括(但不限于)：(1)DNA-DNA或DNA-RNA杂交；(2)标志基因功能的出现或丧失；(3)测定果糖-1，6-二磷酸酶-16.61的转录本的水平；(4)通过免疫学技术或测定生物学活性，来检测基因表达的蛋白产物。上述方法可单用，也可多种方法联合应用。These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) presence or loss of marker gene function; (3) assay of fructose-1,6-bisphosphatase-16.61 transcript (4) detect the protein product expressed by the gene by immunological techniques or measuring biological activity. The above methods can be used alone or in combination with multiple methods.

在第(1)种方法中，杂交所用的探针是与本发明的多核苷酸的任何一部分同源，其长度至少10个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸。此外，探针的长度通常在2000个核苷酸之内，较佳的为1000个核苷酸之内。此处所用的探针通常是在本发明的基因序列信息的基础上化学合成的DNA序列。本发明的基因本身或者片段当然可以用作探针。DNA探针的标记可用放射性同位素，荧光素或酶(如碱性磷酸酶)等。In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probes used here are usually DNA sequences chemically synthesized based on the gene sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

在第(4)种方法中，检测果糖-1，6-二磷酸酶-16.61基因表达的蛋白产物可用免疫学技术如Western印迹法，放射免疫沉淀法，酶联免疫吸附法(ELISA)等。In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the fructose-1,6-bisphosphatase-16.61 gene.

应用PCR技术扩增DNA/RNA的方法(Saiki，et al.Science1985；230：1350-1354)被优选用于获得本发明的基因。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain the full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein. selected and synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

如上所述得到的本发明的基因，或者各种DNA片段等的多核苷酸序列可用常规方法如双脱氧链终止法(Sanger et al.PNAS，1977，74：5463-5467)测定。这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的cDNA序列，测序需反复进行。有时需要测定多个克隆的cDNA序列，才能拼接成全长的cDNA序列。The polynucleotide sequences of the genes of the present invention or various DNA fragments obtained as described above can be determined by conventional methods such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或直接用果糖-1，6-二磷酸酶-16.61编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the fructose-1,6-bisphosphatase-16.61 coding sequence, and producing the polynucleotide by recombinant technology Methods of inventing said polypeptides.

本发明中，编码果糖-1，6-二磷酸酶-16.61的多核苷酸序列可插入到载体中，以构成含有本发明所述多核苷酸的重组载体。术语“载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。在本发明中适用的载体包括但不限于：在细菌中表达的基于T7启动子的表达载体(Rosenberg，et al.Gene，1987，56：125)；在哺乳动物细胞中表达的pMSXND表达载体(Lee and Natbans，J Bio Chem.263：3521，1988)和在昆虫细胞中表达的来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用于构建重组表达载体。表达载体的一个重要特征是通常含有复制起始点、启动子、标记基因和翻译调控元件。In the present invention, the polynucleotide sequence encoding fructose-1,6-bisphosphatase-16.61 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Carriers applicable in the present invention include, but are not limited to: the expression vector based on the T7 promoter expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); the pMSXND expression vector expressed in mammalian cells ( Lee and Natbans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector 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, marker genes, and translational regulatory elements.

本领域的技术人员熟知的方法能用于构建含编码果糖-1，6-二磷酸酶-16.61的DNA序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等(Sambroook，et al.Molecular Cloning，aLaboratory Manual，cold Spring Harbor Laboratory.New York，1989)。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体的P_L启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是DNA表达的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding fructose-1,6-bisphosphatase-16.61 and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage_PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter , LTRs of retroviruses and other promoters known to 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, a transcription terminator, and the like. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性等。In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.

本领域一般技术人员都清楚如何选择适当的载体/转录调控元件(如启动子、增强子等)和选择性标记基因。Those of ordinary skill in the art will know how to select appropriate vectors/transcriptional regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

本发明中，编码果糖-1，6-二磷酸酶-16.61的多核苷酸或含有该多核苷酸的重组载体可转化或转导入宿主细胞，以构成含有该多核苷酸或重组载体的基因工程化宿主细胞。术语“宿主细胞”指原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；细菌细胞如鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇S2或Sf9；动物细胞如CHO、COS或Bowes黑素瘤细胞等。In the present invention, the polynucleotide encoding fructose-1,6-bisphosphatase-16.61 or the recombinant vector containing the polynucleotide can be transformed or transfected into host cells to constitute a genetic engineering gene containing the polynucleotide or recombinant vector cultured host cells. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells, etc.

用本发明所述的DNA序列或含有所述DNA序列的重组载体转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl₂法处理，所用的步骤在本领域众所周知。可供选择的是用MgCl₂。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，或者常规机械方法如显微注射、电穿孔、脂质体包装等。Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the_CaCl2 method using procedures well known in the art. An alternative is to use_MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

通过常规的重组DNA技术，利用本发明的多核苷酸序列可用来表达或生产重组的果糖-1，6-二磷酸酶-16.61(Science，1984；224：1431)。一般来说有以下步骤：The polynucleotide sequence of the present invention can be used to express or produce recombinant fructose-1,6-bisphosphatase-16.61 by conventional recombinant DNA technology (Science, 1984; 224:1431). Generally speaking, there are the following steps:

(1).用本发明的编码人果糖-1，6-二磷酸酶-16.61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). Use the polynucleotide (or variant) encoding human fructose-1,6-bisphosphatase-16.61 of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide ;

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

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

在步骤(2)中，根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。In step (2), the medium used in the culture can be selected from various conventional mediums according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

在步骤(3)中，重组多肽可包被于细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法包括但并不限于：常规的复性处理、蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声波处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。In step (3), the recombinant polypeptide can be encapsulated in the cell, expressed on the cell membrane, or secreted out of the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. These methods include but are not limited to: conventional refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic destruction, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

本发明的多肽以及该多肽的拮抗剂、激动剂和抑制剂可直接用于疾病治疗，例如，可治疗恶性肿瘤、肾上腺缺乏症、皮肤病、各类炎症、HIV感染和免疫性疾病等。The polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used in the treatment of diseases, for example, can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infection and immune diseases.

果糖-1，6-二磷酸酶-16.61(FBPase)(EC 3.1.3.11)是糖原异生的调控酶类，催化果糖-1，6-二磷酸水解产生果糖-6-磷酸。该过程存在于大多数生物的各种代谢途径。Fructose-1,6-bisphosphatase-16.61 (FBPase) (EC 3.1.3.11) is a regulatory enzyme of gluconeogenesis, which catalyzes the hydrolysis of fructose-1,6-bisphosphate to produce fructose-6-phosphate. This process is present in various metabolic pathways of most organisms.

在哺乳动物的果糖-1，6-二磷酸酶-16.61中，一赖氨酸残基与该酶的催化机制密切相关，该残基周围区域的序列高度保守，该特征性的保守序列是形成其活性motif所必需的。该特征序列的表达异常将导致糖代谢紊乱，进而引发相关的疾病.In mammalian fructose-1,6-bisphosphatase-16.61, a lysine residue is closely related to the catalytic mechanism of the enzyme, and the sequence of the region around this residue is highly conserved, and this characteristic conserved sequence is formed Required for its active motif. The abnormal expression of this characteristic sequence will lead to the disorder of glucose metabolism, and then lead to related diseases.

本发明新的多肽在结构与功能上与果糖-1，6-二磷酸酶-16.61有高度的同源性和相似性，并且氨基酸序列中亦含有上述保守的特征性序列模板。本发明新的多肽具有和果糖-1，6-二磷酸酶-16.61相似的生理活性。上述特征性的保守序列的表达异常将导致本发明的含有此motif的新的多肽的功能异常，从而导致糖代谢紊乱，进而引发相关的疾病。一.全身多系统血管硬化1.心脑血管：心绞痛，心肌梗塞，心律失常，冠心病，代谢性心肌病，心力衰竭，心源性休克(主动脉、冠状动脉、心脏微血管)，短暂性脑缺血发作，脑梗塞，腔隙性梗塞，脑出血(脑内动脉)等；2.肾脏血管：肾动脉狭窄，肾动脉栓塞及血栓，小动脉性肾硬化(良性、恶性)，急/慢性肾功能衰竭等；3.肢体外周血管：闭塞性动脉硬化(下肢动脉)，营养不良性皮肤溃疡(皮肤小动脉)等；二.眼科疾病：代谢性白内障，屈光不正，虹膜睫状体炎，眼球运动神经麻痹，视网膜病变(单纯性、增殖性)，虹膜红变，新生血管性青光眼等；三.神经系统疾病：周围神经病(对称性远端性多发性神经病、多发性单神经病、自主神经病)，脊髓病，高渗性昏迷，低血糖性脑病，痴呆，瘫痪等；四.各种感染：皮肤感染(疖、痈、足、体、败血症)，生殖系统感染(真菌性阴道炎、巴氏腺炎)，泌尿系统感染(肾盂肾炎、膀胱炎)等；The novel polypeptide of the present invention has high homology and similarity with fructose-1,6-bisphosphatase-16.61 in structure and function, and the amino acid sequence also contains the above-mentioned conserved characteristic sequence template. The novel polypeptide of the present invention has similar physiological activity to fructose-1,6-bisphosphatase-16.61. The abnormal expression of the above-mentioned characteristic conserved sequence will lead to the abnormal function of the novel polypeptide containing this motif of the present invention, which will lead to the disorder of glucose metabolism, and then lead to related diseases. 1. Systemic multi-system vascular sclerosis 1. Cardiovascular: angina pectoris, myocardial infarction, arrhythmia, coronary heart disease, metabolic cardiomyopathy, heart failure, cardiogenic shock (aorta, coronary artery, cardiac microvascular), transient cerebral infarction Ischemic attack, cerebral infarction, lacunar infarction, cerebral hemorrhage (intracerebral artery), etc.; 2. Renal vessels: renal artery stenosis, renal artery embolism and thrombus, arteriolar nephrosclerosis (benign, malignant), acute/chronic Renal failure, etc.; 3. Peripheral blood vessels of limbs: occlusive arteriosclerosis (lower extremity arteries), dystrophic skin ulcers (skin arterioles), etc.; 2. Eye diseases: metabolic cataract, refractive error, iridocyclitis , oculomotor nerve palsy, retinopathy (simple, proliferative), iris redness, neovascular glaucoma, etc.; 3. Nervous system diseases: peripheral neuropathy (symmetrical distal polyneuropathy, multiple mononeuropathy, autonomic Neuropathy), myelopathy, hyperosmolar coma, hypoglycemic encephalopathy, dementia, paralysis, etc.; 4. Various infections: skin infection (furb, carbuncle, foot, body, sepsis), reproductive system infection (fungal vaginitis, Bartholin's glanditis), urinary system infection (pyelonephritis, cystitis), etc.;

综合上述，本发明的多肽以及该多肽的拮抗剂，激动剂和抑制剂可直接用于多种疾病的诊治，例如心律失常、肾功能衰竭、代谢性白内障、周围神经病、感染等。In summary, the polypeptide of the present invention and its antagonist, agonist and inhibitor can be directly used in the diagnosis and treatment of various diseases, such as arrhythmia, renal failure, metabolic cataract, peripheral neuropathy, infection and so on.

本发明也提供了筛选化合物以鉴定提高(激动剂)或阻遏(拮抗剂)果糖-1，6-二磷酸酶-16.61的药剂的方法。激动剂提高果糖-1，6-二磷酸酶-16.61刺激细胞增殖等生物功能，而拮抗剂阻止和治疗与细胞过度增殖有关的紊乱如各种癌症。例如，能在药物的存在下，将哺乳动物细胞或表达果糖-1，6-二磷酸酶-16.61的膜制剂与标记的果糖-1，6-二磷酸酶-16.61一起培养。然后测定药物提高或阻遏此相互作用的能力。The invention also provides methods of screening compounds to identify agents that increase (agonists) or repress (antagonists) fructose-1,6-bisphosphatase-16.61. Agonists enhance biological functions of fructose-1,6-bisphosphatase-16.61 such as stimulation of cell proliferation, while antagonists prevent and treat disorders associated with excessive cell proliferation such as various cancers. For example, mammalian cells or membrane preparations expressing fructose-1,6-bisphosphatase-16.61 can be incubated with labeled fructose-1,6-bisphosphatase-16.61 in the presence of the drug. The ability of the drug to enhance or repress this interaction is then determined.

果糖-1，6-二磷酸酶-16.61的拮抗剂包括筛选出的抗体、化合物、受体缺失物和类似物等。果糖-1，6-二磷酸酶-16.61的拮抗剂可以与果糖-1，6-二磷酸酶-16.61结合并消除其功能，或是抑制该多肽的产生，或是与该多肽的活性位点结合使该多肽不能发挥生物学功能。Antagonists of fructose-1,6-bisphosphatase-16.61 include screened antibodies, compounds, receptor deletions and analogs. Antagonists of fructose-1,6-bisphosphatase-16.61 can bind to fructose-1,6-bisphosphatase-16.61 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide Binding renders the polypeptide incapable of biological function.

在筛选作为拮抗剂的化合物时，可以将果糖-1，6-二磷酸酶-16.61加入生物分析测定中，通过测定化合物对果糖-1，6-二磷酸酶-16.61和其受体之间相互作用的影响来确定化合物是否是拮抗剂。用上述筛选化合物的同样方法，可以筛选出起拮抗剂作用的受体缺失物和类似物。能与果糖-1，6-二磷酸酶-16.61结合的多肽分子可通过筛选由各种可能组合的氨基酸结合于固相物组成的随机多肽库而获得。筛选时，一般应对果糖-1，6-二磷酸酶-16.61分子进行标记。In screening compounds as antagonists, fructose-1,6-bisphosphatase-16.61 can be added to bioanalytical assays by measuring the interaction between fructose-1,6-bisphosphatase-16.61 and its receptors. effect to determine whether a compound is an antagonist. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. The polypeptide molecule capable of binding to fructose-1,6-bisphosphatase-16.61 can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the fructose-1,6-bisphosphatase-16.61 molecule should generally be labeled.

本发明提供了用多肽，及其片段、衍生物、类似物或它们的细胞作为抗原以生产抗体的方法。这些抗体可以是多克隆抗体或单克隆抗体。本发明还提供了针对果糖-1，6-二磷酸酶-16.61抗原决定簇的抗体。这些抗体包括(但不限于)：多克隆抗体、单克隆抗体、嵌合抗体、单链抗体、Fab片段和Fab表达文库产生的片段。The present invention provides methods for producing antibodies using polypeptides, fragments, derivatives, analogs thereof, or cells thereof as antigens. These antibodies can be polyclonal or monoclonal. The present invention also provides antibodies against the epitope of fructose-1,6-bisphosphatase-16.61. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

多克隆抗体的生产可用果糖-1，6-二磷酸酶-16.61直接注射免疫动物(如家兔，小鼠，大鼠等)的方法得到，多种佐剂可用于增强免疫反应，包括但不限于弗氏佐剂等。制备果糖-1，6-二磷酸酶-16.61的单克隆抗体的技术包括但不限于杂交瘤技术(Kohler and Milstein.Nature，1975，256：495-497)，三瘤技术，人B-细胞杂交瘤技术，EBV-杂交瘤技术等。将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产(Morrison et al，PNAS，1985，81：6851)。而已有的生产单链抗体的技术(U.S.Pat No.4946778)也可用于生产抗果糖-1，6-二磷酸酶-16.61的单链抗体。The production of polyclonal antibodies can be obtained by direct injection of fructose-1,6-bisphosphatase-16.61 into immunized animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance the immune response, including but not Limited to Freund's adjuvant, etc. Preparation of fructose-1,6-bisphosphatase-16.61 monoclonal antibody technology includes but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), triple tumor technology, human B-cell hybridization tumor technology, EBV-hybridoma technology, etc. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81:6851). And the existing technology (U.S.Pat No.4946778) of producing single-chain antibody can also be used for producing the single-chain antibody of anti-fructose-1,6-bisphosphatase-16.61.

抗果糖-1，6-二磷酸酶-16.61的抗体可用于免疫组织化学技术中，检测活检标本中的果糖-1，6-二磷酸酶-16.61。Antibodies against fructose-1,6-bisphosphatase-16.61 can be used in immunohistochemical techniques to detect fructose-1,6-bisphosphatase-16.61 in biopsy specimens.

与果糖-1，6-二磷酸酶-16.61结合的单克隆抗体也可用放射性同位素标记，注入体内可跟踪其位置和分布。这种放射性标记的抗体可作为一种非创伤性诊断方法用于肿瘤细胞的定位和判断是否有转移。Monoclonal antibodies combined with fructose-1,6-bisphosphatase-16.61 can also be labeled with radioactive isotopes, and injected into the body to track its location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

抗体还可用于设计针对体内某一特殊部位的免疫毒素。如果糖-1，6-二磷酸酶-16.61高亲和性的单克隆抗体可与细菌或植物毒素(如白喉毒素，蓖麻蛋白，红豆碱等)共价结合。一种通常的方法是用巯基交联剂如SPDP，攻击抗体的氨基，通过二硫键的交换，将毒素结合于抗体上，这种杂交抗体可用于杀灭果糖-1，6-二磷酸酶-16.61阳性的细胞。Antibodies can also be used to design immunotoxins that target a particular site in the body. If sugar-1,6-bisphosphatase-16.61 high-affinity monoclonal antibody can be covalently combined with bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill fructose-1,6-bisphosphatase -16.61 positive cells.

本发明中的抗体可用于治疗或预防与果糖-1，6-二磷酸酶-16.61相关的疾病。给予适当剂量的抗体可以刺激或阻断果糖-1，6-二磷酸酶-16.61的产生或活性。The antibody of the present invention can be used to treat or prevent diseases related to fructose-1,6-bisphosphatase-16.61. Administration of appropriate doses of the antibody can stimulate or block the production or activity of fructose-1,6-bisphosphatase-16.61.

本发明还涉及定量和定位检测果糖-1，6-二磷酸酶-16.61水平的诊断试验方法。这些试验是本领域所熟知的，且包括FISH测定和放射免疫测定。试验中所检测的果糖-1，6-二磷酸酶-16.61水平，可以用作解释果糖-1，6-二磷酸酶-16.61在各种疾病中的重要性和用于诊断果糖-1，6-二磷酸酶-16.61起作用的疾病。The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of fructose-1,6-bisphosphatase-16.61. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of fructose-1,6-bisphosphatase-16.61 detected in the test can be used to explain the importance of fructose-1,6-bisphosphatase-16.61 in various diseases and to diagnose fructose-1,6 - Diseases in which bisphosphatase-16.61 plays a role.

本发明的多肽还可用作肽谱分析，例如，多肽可用物理的、化学或酶进行特异性切割，并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分析。The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cut physically, chemically or enzymatically, and then subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis, and better mass spectrometry analysis.

编码果糖-1，6-二磷酸酶-16.61的多核苷酸也可用于多种治疗目的。基因治疗技术可用于治疗由于果糖-1，6-二磷酸酶-16.61的无表达或异常/无活性表达所致的细胞增殖、发育或代谢异常。重组的基因治疗载体(如病毒载体)可设计用于表达变异的果糖-1，6-二磷酸酶-16.61，以抑制内源性的果糖-1，6-二磷酸酶-16.61活性。例如，一种变异的果糖-1，6-二磷酸酶-16.61可以是缩短的、缺失了信号传导功能域的果糖-1，6-二磷酸酶-16.61，虽可与下游的底物结合，但缺乏信号传导活性。因此重组的基因治疗载体可用于治疗果糖-1，6-二磷酸酶-16.61表达或活性异常所致的疾病。来源于病毒的表达载体如逆转录病毒、腺病毒、腺病毒相关病毒、单纯疱疹病毒、细小病毒等可用于将编码果糖-1，6-二磷酸酶-16.61的多核苷酸转移至细胞内。构建携带编码果糖-1，6-二磷酸酶-16.61的多核苷酸的重组病毒载体的方法可见于已有文献(Sambrook，et al.)。另外重组编码果糖-1，6-二磷酸酶-16.61的多核苷酸可包装到脂质体中转移至细胞内。Polynucleotides encoding fructose-1,6-bisphosphatase-16.61 are also useful for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression or abnormal/inactive expression of fructose-1,6-bisphosphatase-16.61. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant fructose-1,6-bisphosphatase-16.61 to inhibit the activity of endogenous fructose-1,6-bisphosphatase-16.61. For example, a variant of fructose-1,6-bisphosphatase-16.61 can be a shortened fructose-1,6-bisphosphatase-16.61 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of fructose-1,6-bisphosphatase-16.61. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding fructose-1,6-bisphosphatase-16.61 into cells. The method for constructing a recombinant viral vector carrying a polynucleotide encoding fructose-1,6-bisphosphatase-16.61 can be found in existing literature (Sambrook, et al.). In addition, the recombinant polynucleotide encoding fructose-1,6-bisphosphatase-16.61 can be packaged into liposomes and transferred into cells.

多核苷酸导入组织或细胞内的方法包括：将多核苷酸直接注入到体内组织中；或在体外通过载体(如病毒、噬菌体或质粒等)先将多核苷酸导入细胞中，再将细胞移植到体内等。Methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.), and then transplanting the cells Wait in the body.

抑制果糖-1，6-二磷酸酶-16.61 mRNA的寡核苷酸(包括反义RNA和DNA)以及核酶也在本发明的范围之内。核酶是一种能特异性分解特定RNA的酶样RNA分子，其作用机制是核酶分子与互补的靶RNA特异性杂交后进行核酸内切作用。反义的RNA和DNA及核酶可用已有的任何RNA或DNA合成技术获得，如固相磷酸酰胺化学合成法合成寡核苷酸的技术已广泛应用。反义RNA分子可通过编码该RNA的DNA序列在体外或体内转录获得。这种DNA序列已整合到载体的RNA聚合酶启动子的下游。为了增加核酸分子的稳定性，可用多种方法对其进行修饰，如增加两侧的序列长度，核糖核苷之间的连接应用磷酸硫酯键或肽键而非磷酸二酯键。Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit fructose-1,6-bisphosphatase-16.61 mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

编码果糖-1，6-二磷酸酶-16.61的多核苷酸可用于与果糖-1，6-二磷酸酶-16.61的相关疾病的诊断。编码果糖-1，6-二磷酸酶-16.61的多核苷酸可用于检测果糖-1，6-二磷酸酶-16.61的表达与否或在疾病状态下果糖-1，6-二磷酸酶-16.61的异常表达。如编码果糖-1，6-二磷酸酶-16.61的DNA序列可用于对活检标本进行杂交以判断果糖-1，6-二磷酸酶-16.61的表达状况。杂交技术包括Southern印迹法，Northern印迹法、原位杂交等。这些技术方法都是公开的成熟技术，相关的试剂盒都可从商业途径得到。本发明的多核苷酸的一部分或全部可作为探针固定在微阵列(Microarray)或DNA芯片(又称为“基因芯片”)上，用于分析组织中基因的差异表达分析和基因诊断。用果糖-1，6-二磷酸酶-16.61特异的引物进行RNA-聚合酶链反应(RT-PCR)体外扩增也可检测果糖-1，6-二磷酸酶-16.61的转录产物。The polynucleotide encoding fructose-1,6-bisphosphatase-16.61 can be used for the diagnosis of diseases related to fructose-1,6-bisphosphatase-16.61. The polynucleotide encoding fructose-1,6-bisphosphatase-16.61 can be used to detect the expression of fructose-1,6-bisphosphatase-16.61 or fructose-1,6-bisphosphatase-16.61 in disease state abnormal expression. For example, the DNA sequence encoding fructose-1,6-bisphosphatase-16.61 can be used for hybridization of biopsy specimens to determine the expression status of fructose-1,6-bisphosphatase-16.61. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. Fructose-1,6-bisphosphatase-16.61 transcripts can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for fructose-1,6-bisphosphatase-16.61.

检测果糖-1，6-二磷酸酶-16.61基因的突变也可用于诊断果糖-1，6-二磷酸酶-16.61相关的疾病。果糖-1，6-二磷酸酶-16.61突变的形式包括与正常野生型果糖-1，6-二磷酸酶-16.61 DNA序列相比的点突变、易位、缺失、重组和其它任何异常等。可用已有的技术如Southern印迹法、DNA序列分析、PCR和原位杂交检测突变。另外，突变有可能影响蛋白的表达，因此用Northern印迹法、Western印迹法可间接判断基因有无突变。Detection of mutations in the fructose-1,6-bisphosphatase-16.61 gene can also be used to diagnose fructose-1,6-bisphosphatase-16.61-related diseases. Mutant forms of fructose-1,6-bisphosphatase-16.61 include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type fructose-1,6-bisphosphatase-16.61 DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

本发明的序列对染色体鉴定也是有价值的。该序列会特异性地针对某条人染色体具体位置且并可以与其杂交。目前，需要鉴定染色体上的各基因的具体位点。现在，只有很少的基于实际序列数据(重复多态性)的染色体标记物可用于标记染色体位置。根据本发明，为了将这些序列与疾病相关基因相关联，其重要的第一步就是将这些DNA序列定位于染色体上。The sequences of the invention are also valuable for chromosome identification. The sequence will be specific for a particular location on a human chromosome and can hybridize thereto. Currently, there is a need to identify the specific site of each gene on the chromosome. Currently, only a few chromosomal markers based on actual sequence data (repeat polymorphisms) are available to mark chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on chromosomes.

简而言之，根据cDNA制备PCR引物(优选15-35bp)，可以将序列定位于染色体上。然后，将这些引物用于PCR筛选含各条人染色体的体细胞杂合细胞。只有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

体细胞杂合细胞的PCR定位法，是将DNA定位到具体染色体的快捷方法。使用本发明的寡核苷酸引物，通过类似方法，可利用一组来自特定染色体的片段或大量基因组克隆而实现亚定位。可用于染色体定位的其它类似策略包括原位杂交、用标记的流式分选的染色体预筛选和杂交预选，从而构建染色体特异的cDNA库。The PCR mapping method of somatic heterozygous cells is a quick method to locate DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved using a set of fragments from a particular chromosome or a large number of genomic clones by a similar method. Other similar strategies that can be used for chromosome mapping include in situ hybridization, chromosome prescreening by flow sorting with markers, and hybridization preselection to construct chromosome-specific cDNA libraries.

将cDNA克隆与中期染色体进行荧光原位杂交(FISH)，可以在一个步骤中精确地进行染色体定位。此技术的综述，参见Verma等，Human Chromosomes：a Manualof Basic Techniques，Pergamon Press，New York(1988)。Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal mapping in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

一旦序列被定位到准确的染色体位置，此序列在染色体上的物理位置就可以与基因图数据相关联。这些数据可见于例如，V.Mckusick，Mendelian Inheritancein Man(可通过与Johns Hopkins University Welch Medical Library联机获得)。然后可通过连锁分析，确定基因与业已定位到染色体区域上的疾病之间的关系。Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

接着，需要测定患病和未患病个体间的cDNA或基因组序列差异。如果在一些或所有的患病个体中观察到某突变，而该突变在任何正常个体中未观察到，则该突变可能是疾病的病因。比较患病和未患病个体，通常涉及首先寻找染色体中结构的变化，如从染色体水平可见的或用基于cDNA序列的PCR可检测的缺失或易位。根据目前的物理作图和基因定位技术的分辨能力，被精确定位至与疾病有关的染色体区域的cDNA，可以是50至500个潜在致病基因间之一种(假定1兆碱基作图分辨能力和每20kb对应于一个基因)。Next, the cDNA or genome sequence differences between affected and non-affected individuals need to be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping techniques, the cDNA that is pinpointed to a disease-associated chromosomal region can be one of 50 to 500 potential disease-causing genes (assuming 1 megabase mapping resolution capacity and each 20kb corresponds to a gene).

可以将本发明的多肽、多核苷酸及其模拟物、激动剂、拮抗剂和抑制剂与合适的药物载体组合后使用。这些载体可以是水、葡萄糖、乙醇、盐类、缓冲液、甘油以及它们的组合。组合物包含安全有效量的多肽或拮抗剂以及不影响药物效果的载体和赋形剂。这些组合物可以作为药物用于疾病治疗。The polypeptides, polynucleotides and their mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with appropriate pharmaceutical carriers. These carriers can be water, dextrose, ethanol, salts, buffers, glycerol and combinations thereof. The composition contains safe and effective doses of polypeptides or antagonists as well as carriers and excipients that do not affect the drug effect. These compositions can be used as medicine for disease treatment.

本发明还提供含有一种或多种容器的药盒或试剂盒，容器中装有一种或多种本发明的药用组合物成分。与这些容器一起，可以有由制造、使用或销售药品或生物制品的政府管理机构所给出的指示性提示，该提示反映出生产、使用或销售的政府管理机构许可其在人体上施用。此外，本发明的多肽可以与其它的治疗化合物结合使用。The invention also provides kits or kits comprising one or more containers containing one or more ingredients of the pharmaceutical compositions of the invention. Along with these containers, there may be an indicative notice given by the governmental regulatory agency that manufactures, uses or sells the drug or biological product reflecting its approval for human administration by the governmental regulatory agency that manufactures, uses or sells the drug or biological product. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

药物组合物可以以方便的方式给药，如通过局部、静脉内、腹膜内、肌内、皮下、鼻内或皮内的给药途径。果糖-1，6-二磷酸酶-16.61以有效地治疗和/或预防具体的适应症的量来给药。施用于患者的果糖-1，6-二磷酸酶-16.61的量和剂量范围将取决于许多因素，如给药方式、待治疗者的健康条件和诊断医生的判断。The pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration. Fructose-1,6-bisphosphatase-16.61 is administered in an amount effective to treat and/or prevent the particular indication. The amount and dosage range of fructose-1,6-bisphosphatase-16.61 administered to a patient will depend on many factors, such as the mode of administration, the health condition of the person to be treated, and the judgment of the diagnosing physician.

                            附图说明Description of 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 present invention, but not to limit the scope of the present invention defined by the claims.

图1是本发明果糖-1，6-二磷酸酶-16.61在2-67共66个氨基酸和结构域MOTIF的氨基酸序列同源性比较图。上方序列是果糖-1，6-二磷酸酶-16.61，下方序列是结构域MOTIF。“|”和“:”及“.”表示在两个序列间同一氨基酸出现的概率依次减少。Fig. 1 is a comparison diagram of amino acid sequence homology of fructose-1,6-bisphosphatase-16.61 of the present invention, including 66 amino acids at 2-67 and the domain MOTIF. The upper sequence is fructose-1,6-bisphosphatase-16.61 and the lower sequence is domain MOTIF. "|" and ":" and "." indicate that the probability of the same amino acid appearing between the two sequences decreases in turn.

图2为分离的果糖-1，6-二磷酸酶-16.61的聚丙烯酰胺凝胶电泳图(SDS-PAGE)。16.61kDa为蛋白质的分子量。箭头所指为分离出的蛋白条带。Figure 2 is the polyacrylamide gel electrophoresis pattern (SDS-PAGE) of isolated fructose-1,6-bisphosphatase-16.61. 16.61kDa is the molecular weight of the protein. Arrows indicate isolated protein bands.

                           具体实施方式 Detailed ways

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 HarborLaboratory 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：果糖-1，6-二磷酸酶-16.61的克隆Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Example 1: Cloning of fructose-1,6-bisphosphatase-16.61

用异硫氰酸胍/酚/氯仿一步法提取人胎脑总RNA。用Quik mRNA Isolation Kit(Qiegene公司产品)从总RNA中分离poly(A)mRNA。2ug poly(A)mRNA经逆转录形成cDNA。用Smart cDNA克隆试剂盒(购自Clontech)将cDNA片段定向插入到pBSK(+)载体(Clontech公司产品)的多克隆位点上，转化DH5α，细菌形成cDNA文库。用Dyeterminate cycle reaction sequencing kit(Perkin-Elmer公司产品)和ABI 377自动测序仪(Perkin-Elmer公司)测定所有克隆的5′和3′末端的序列。将测定的cDNA序列与已有的公共DNA序列数据库(Genebank)进行比较，结果发现其中一个克隆4676b09的cDNA序列为新的DNA。通过合成一系列引物对该克隆所含的插入cDNA片段进行双向测定。结果表明，4676b09克隆所含的全长cDNA为1231bp(如<210>1所示)，从第55bp至510bp有一个264bp的开放阅读框架(ORF)，编码一个新的蛋白质(如<210>2所示)。我们将此克隆命名为pBS-4676b09，编码的蛋白质命名为果糖-1，6-二磷酸酶-16.61。实施例2：cDNA克隆的结构域分析Total RNA was extracted from human fetal brain by one-step method of guanidine isothiocyanate/phenol/chloroform. Poly(A) mRNA was isolated from total RNA with Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. The cDNA fragment was directional inserted into the multiple cloning site of the pBSK (+) vector (product of Clontech) using the Smart cDNA cloning kit (purchased from Clontech), transformed into DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined with Dyeterminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 4676b09, was a new DNA. The insert cDNA fragment contained in this clone was determined bidirectionally by synthesizing a series of primers. The results showed that the full-length cDNA contained in the 4676b09 clone was 1231bp (shown as <210>1), and there was an open reading frame (ORF) of 264bp from 55bp to 510bp, encoding a new protein (shown as <210>2 shown). We named this clone pBS-4676b09, and named the encoded protein fructose-1,6-bisphosphatase-16.61. Example 2: Domain analysis of cDNA clones

将本发明的果糖-1，6-二磷酸酶-16.61的序列及其编码的蛋白序列，用GCG中的profile scan程序(Basiclocal Alignment search tool)[Altschul，SF et al.J.Mol.Biol.1990；215：403-10]，在prosite等数据库进行结构域分析。本发明的果糖-1，6-二磷酸酶-16.61与结构域MOTIF有同源，同源结果示于图1，同源率为36％，得分为14.87；阈值为16.44。实施例3：用RT-PCR方法克隆编码果糖-1，6-二磷酸酶-16.61的基因With the fructose-1 of the present invention, the sequence of 6-bisphosphatase-16.61 and its coded protein sequence, use the profile scan program (Basiclocal Alignment search tool) in GCG [Altschul, SF et al.J.Mol.Biol. 1990; 215: 403-10], domain analysis in databases such as prosite. The fructose-1,6-bisphosphatase-16.61 of the present invention has homology with the domain MOTIF, the homology result is shown in Figure 1, the homology rate is 36%, the score is 14.87; the threshold is 16.44. Embodiment 3: Cloning the gene encoding fructose-1,6-bisphosphatase-16.61 by RT-PCR method

用胎脑细胞总RNA为模板，以oligo-dT为引物进行逆转录反应合成cDNA，用Qiagene的试剂盒纯化后，用下列引物进行PCR扩增：The total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer to carry out reverse transcription reaction to synthesize cDNA. After purification with a Qiagene kit, PCR amplification was performed with the following primers:

Primer1：5’-CATCCTGAGAACTGAAATTGATCGC-3’(<210>3)Primer1: 5'-CATCCTGAGAACTGAAATTGATCGC-3'(<210>3)

Primer2：5’-ATAAAATTTTTGAATTTATGTTCAA-3’(<210>4)Primer2: 5'-ATAAAATTTTTGAATTTATGTTCAA-3'(<210>4)

Primer1为位于<210>1的5’端的第1bp开始的正向序列；Primer1 is the forward sequence starting from the 1bp at the 5' end of <210>1;

Primer2为<210>1的中的3’端反向序列。Primer2 is the 3' reverse sequence in <210>1.

扩增反应的条件：在50μl的反应体积中含有50mmol/L KCl，10mmol/LTris-Cl，(pH8.5)，1.5mmol/L MgCl₂，200μmol/L dNTP，10pmol引物，1U的Taq DNA聚合酶(Clontech公司产品)。在PE9600型DNA热循环仪(Perkin-Elmer公司)上按下列条件反应25个周期：94℃30sec；55℃30sec；72℃2min。在RT-PCR时同时设β-actin为阳性对照和模板空白为阴性对照。扩增产物用QIAGEN公司的试剂盒纯化，用TA克隆试剂盒连接到pCR载体上(Invitrogen公司产品)。DNA序列分析结果表明PCR产物的DNA序列与<210>1所示的1-1231bp完全相同。实施例4：Northern印迹法分析果糖-1，6-二磷酸酶-16.61基因的表达：The conditions of the amplification reaction: 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl₂ , 200μmol/L dNTP, 10pmol primers, 1U Taq DNA polymer in a reaction volume of 50μl Enzyme (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer), the reaction was performed for 25 cycles under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C for 2 min. During RT-PCR, β-actin was set as positive control and template blank was set as negative control. The amplified product was purified with a kit from QIAGEN, and connected to a pCR vector (product of Invitrogen) with a TA cloning kit. The results of DNA sequence analysis showed that the DNA sequence of the PCR product was exactly the same as 1-1231bp indicated by <210>1. Example 4: Northern blot analysis of the expression of fructose-1,6-bisphosphatase-16.61 gene:

用一步法提取总RNA[Anal.Biochem 1987，162，156-159]。该法包括酸性硫氰酸胍苯酚-氯仿抽提。即用4M异硫氰酸胍-25mM柠檬酸钠，0.2M乙酸钠(pH4.0)对组织进行匀浆，加入1倍体积的苯酚和1/5体积的氯仿-异戊醇(49∶1)，混合后离心。吸出水相层，加入异丙醇(0.8体积)并将混合物离心得到RNA沉淀。将得到的RNA沉淀用70％乙醇洗涤，干燥并溶于水中。用20μg RNA，在含20mM 3-(N-吗啉代)丙磺酸(pH7.0)-5mM乙酸钠-1mM EDTA-2.2M甲醛的1.2％琼脂糖凝胶上进行电泳。然后转移至硝酸纤维素膜上。用α-³²P dATP通过随机引物法制备³²P-标记的DNA探针。所用的DNA探针为图1所示的PCR扩增的果糖-1，6-二磷酸酶-16.61编码区序列(55bp至510bp)。将32P-标记的探针(约2×10⁶cpm/ml)与转移了RNA的硝酸纤维素膜在一溶液中于42℃杂交过夜，该溶液包含50％甲酰胺-25mM KH₂PO₄(pH7.4)-5×SSC-5×Denhardt’s溶液和200μg/ml鲑精DNA。杂交之后，将滤膜在1×SSC-0.1％SDS中于55℃洗30min。然后，用Phosphor Imager进行分析和定量。实施例5：重组果糖-1，6-二磷酸酶-16.61的体外表达、分离和纯化Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane.³² P-labeled DNA probes were prepared by the random primer method using α-³² P dATP. The DNA probe used is the fructose-1,6-bisphosphatase-16.61 coding region sequence (55bp to 510bp) amplified by PCR shown in FIG. 1 . The 32P-labeled probe (approximately 2×10⁶ cpm/ml) was hybridized to the RNA-transferred nitrocellulose membrane at 42° C. overnight in a solution containing 50% formamide-25 mM KH₂ PO₄ ( pH7.4)-5*SSC-5*Denhardt's solution and 200 μg/ml salmon sperm DNA. After hybridization, the filters were washed in 1×SSC-0.1% SDS at 55° C. for 30 min. Then, analysis and quantification were performed with Phosphor Imager. Example 5: In vitro expression, isolation and purification of recombinant fructose-1,6-bisphosphatase-16.61

根据<210>1和图1所示的编码区序列，设计出一对特异性扩增引物，序列如下：According to <210>1 and the sequence of the coding region shown in Figure 1, a pair of specific amplification primers were designed with the sequence as follows:

Primer3：5’-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3’(<210>5)Primer3: 5'-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3'(<210>5)

Primer4：5’-CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3’(<210>6)Primer4: 5'-CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3'(<210>6)

此两段引物的5’端分别含有NdeI和EcoRI酶切位点，其后分别为目的基因5’端和3’端的编码序列，NdeI和EcoRI酶切位点相应于表达载体质粒pET 28b(+)(Novagen公司产品，Cat.No.69865.3)上的选择性内切酶位点。以含有全长目的基因的pBS-4676b09质粒为模板，进行PCR反应。PCR反应条件为：总体积50μl中含pBS-4676b09质粒10pg、引物Primer-3和Primer-4分别为10pmol、Advantage polymerase Mix(Clontech公司产品)1μl。循环参数：94℃20s，60℃30s，68℃2min，共25个循环。用NdeI和EcoRI分别对扩增产物和质粒pET-28(+)进行双酶切，分别回收大片段，并用T4连接酶连接。连接产物转化用氯化钙法大肠杆细菌DH5α，在含卡那霉素(终浓度30μg/ml)的LB平板培养过夜后，用菌落PCR方法筛选阳性克隆，并进行测序。挑选序列正确的阳性克隆(pET-4676b09)用氯化钙法将重组质粒转化大肠杆菌BL21(DE3)plySs(Novagen公司产品)。在含卡那霉素(终浓度30μg/ml)的LB液体培养基中，宿主菌BL21(pET-4676b09)在37℃培养至对数生长期，加入IPTG至终浓度1mmol/L，继续培养5小时。离心收集菌体，经超声波破菌，离心收集上清，用能与6个组氨酸(6His-Tag)结合的亲和层析柱His.Bind Quick Cartridge(Novagen公司产品)进行层析，得到了纯化的目的蛋白果糖-1，6-二磷酸酶-16.61。经SDS-PAGE电泳，在16.61kDa处得到一单一的条带(图2)。将该条带转移至PVDF膜上用Edams水解法进行N-端氨基酸序列分析，结果N-端15个氨基酸与<210>2所示的N-端15个氨基酸残基完全相同。实施例6抗果糖-1，6-二磷酸酶-16.61抗体的产生The 5' ends of these two primers contain NdeI and EcoRI restriction sites respectively, followed by the coding sequences of the 5' and 3' ends of the target gene respectively, and the NdeI and EcoRI restriction sites correspond to the expression vector plasmid pET 28b(+ ) (Novagen company product, Cat.No.69865.3) on the selective endonuclease site. The pBS-4676b09 plasmid containing the full-length target gene was used as a template for PCR reaction. The PCR reaction conditions were as follows: 10 pg of pBS-4676b09 plasmid in a total volume of 50 μl, 10 pmol of primers Primer-3 and Primer-4 respectively, and 1 μl of Advantage polymerase Mix (product of Clontech Company). Cycle parameters: 94°C for 20s, 60°C for 30s, 68°C for 2min, a total of 25 cycles. The amplified product and plasmid pET-28(+) were digested with NdeI and EcoRI respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Escherichia coli DH5α by the calcium chloride method. After culturing overnight on an LB plate containing kanamycin (final concentration 30 μg/ml), positive clones were screened by colony PCR and sequenced. A positive clone (pET-4676b09) with the correct sequence was selected to transform the recombinant plasmid into Escherichia coli BL21(DE3)plySs (product of Novagen) by calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg/ml), the host strain BL21 (pET-4676b09) was cultured at 37°C to the logarithmic growth phase, and IPTG was added to the final concentration of 1 mmol/L, and the culture was continued for 5 Hour. Bacteria were collected by centrifugation, bacteria were destroyed by ultrasonic waves, supernatant was collected by centrifugation, and the affinity chromatography column His.Bind Quick Cartridge (product of Novagen Company) capable of binding to 6 histidines (6His-Tag) was used for chromatography to obtain The purified target protein fructose-1,6-bisphosphatase-16.61 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 16.61kDa (Figure 2). Transfer the band to PVDF membrane and analyze the N-terminal amino acid sequence by Edams hydrolysis method. As a result, the N-terminal 15 amino acid residues are completely identical to the N-terminal 15 amino acid residues indicated by <210>2. Example 6 Production of anti-fructose-1,6-bisphosphatase-16.61 antibody

用多肽合成仪(PE公司产品)合成下述果糖-1，6-二磷酸酶-16.61特异性的多肽：Synthesize the following fructose-1,6-bisphosphatase-16.61 specific polypeptides with a polypeptide synthesizer (product of PE company):

NH2-Met-Leu-Cys-His-Leu-Gln-Arg-Met-Val-Ser-Glu-Gln-Cys-His-Leu-COOH(<210>7)。将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合，方法参见：Avrameas，et al.Immunochemistry，1969；6：43。用4mg上述血蓝蛋白多肽复合物加上完全弗氏佐剂免疫家兔，15天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂加强免疫一次。采用经15μg/ml牛血清白蛋白多肽复合物包被的滴定板做ELISA测定兔血清中抗体的滴度。用蛋白A-Sepharose从抗体阳性的家兔血清中分离总IgG。将多肽结合于溴化氰活化的Sepharose4B柱上，用亲和层析法从总IgG中分离抗多肽抗体。免疫沉淀法证明纯化的抗体可特异性地与果糖-1，6-二磷酸酶-16.61结合。实施例7：本发明的多核苷酸片段用作杂交探针的应用NH2-Met-Leu-Cys-His-Leu-Gln-Arg-Met-Val-Ser-Glu-Gln-Cys-His-Leu-COOH (<210>7). The polypeptide is coupled with hemocyanin and bovine serum albumin respectively to form a complex. For the method, see: Avrameas, et al. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the above-mentioned hemocyanin polypeptide complex plus complete Freund's adjuvant, and then boosted with the hemocyanin polypeptide complex plus incomplete Freund's adjuvant once 15 days later. The titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 μg/ml bovine serum albumin-polypeptide complex. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a Sepharose4B column activated by cyanogen bromide, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation proved that the purified antibody could specifically bind to fructose-1,6-bisphosphatase-16.61. Embodiment 7: the application of polynucleotide fragment of the present invention as hybridization probe

从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的用途，如用该探针可与不同来源的正常组织或病理组织的基因组或cDNA文库杂交以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理组织细胞中的表达是否异常。Select suitable oligonucleotide fragments from the polynucleotides of the present invention to be used as hybridization probes in many ways, such as using the probes to hybridize with genome or cDNA libraries of normal tissues or pathological tissues from different sources to Identify whether it contains the polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence, and further use the probe to detect the presence of the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology Whether the expression in tissue cells is abnormal.

本实施例的目的是从本发明的多核苷酸<210>1中挑选出合适的寡核苷酸片段用作杂交探针，并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核苷酸序列或其同源的多核苷酸序列。滤膜杂交方法包括斑点印迹法、Southern印迹法、Northern印迹法和复印方法等，它们都是将待测的多核苷酸样品固定在滤膜上后使用基本相同的步骤杂交。这些相同的步骤是：固定了样品的滤膜首先用不含探针的杂交缓冲液进行预杂交，以使滤膜上样品的非特异性的结合部位被载体和合成的多聚物所饱和。然后预杂交液被含有标记探针的杂交缓冲液替换，并保温使探针与靶核酸杂交。杂交步骤之后，未杂交上的探针被一系列洗膜步骤除掉。本实施例利用较高强度的洗膜条件(如较低盐浓度和较高的温度)，以使杂交背景降低且只保留特异性强的信号。本实施例选用的探针包括两类：第一类探针是完全与本发明的多核苷酸<210>1相同或互补的寡核苷酸片段；第二类探针是部分与本发明的多核苷酸<210>1相同或互补的寡核苷酸片段。本实施例选用斑点印迹法将样品固定在滤膜上，在较高强度的的洗膜条件下，第一类探针与样品的杂交特异性最强而得以保留。一、探针的选用The purpose of this example is to select a suitable oligonucleotide fragment from the polynucleotide <210>1 of the present invention as a hybridization probe, and use the filter hybridization method to identify whether some tissues contain the polynucleotide of the present invention sequence or its homologous polynucleotide sequence. Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, all of which use basically the same steps to hybridize after immobilizing the polynucleotide sample to be tested on the filter membrane. These same steps are: the sample-immobilized filter is first prehybridized with probe-free hybridization buffer, so that the non-specific binding sites of the sample on the filter are saturated with the support and synthetic polymer. The prehybridization solution is then replaced with hybridization buffer containing labeled probes and incubated to allow the probes to hybridize to the target nucleic acid. After the hybridization step, unhybridized probes are removed by a series of washing steps. In this embodiment, higher strength membrane washing conditions (such as lower salt concentration and higher temperature) are used to reduce the background of hybridization and retain only highly specific signals. The probes selected in this embodiment include two types: the first type of probe is an oligonucleotide fragment that is completely identical or complementary to the polynucleotide <210>1 of the present invention; the second type of probe is partially identical to the polynucleotide of the present invention Polynucleotide <210>1 Identical or complementary oligonucleotide fragments. In this embodiment, the dot blot method is used to immobilize the sample on the filter membrane. Under the condition of relatively high intensity washing membrane, the hybridization specificity of the first type of probe to the sample is the strongest and thus retained. 1. Selection of probes

从本发明的多核苷酸<210>1中选择寡核苷酸片段用作杂交探针，应遵循以下原则和需要考虑的几个方面：1，探针大小优选范围为18-50个核苷酸；2，GC含量为30％-70％，超过则非特异性杂交增加；3，探针内部应无互补区域；4，符合以上条件的可作为初选探针，然后进一步作计算机序列分析，包括将该初选探针分别与其来源序列区域(即<210>1)和其它已知的基因组序列及其互补区进行同源性比较，若与非靶分子区域的同源性大于85％或者有超过15个连续碱基完全相同，则该初选探针一般就不应该使用；5，初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。完成以上各方面的分析后挑选并合成以下二个探针：Selecting oligonucleotide fragments from the polynucleotide <210>1 of the present invention as a hybridization probe should follow the following principles and several aspects to be considered: 1. The preferred range of probe size is 18-50 nucleosides 2. GC content is 30%-70%, if it exceeds, non-specific hybridization will increase; 3. There should be no complementary region inside the probe; 4. Those that meet the above conditions can be used as primary probes, and then further computer sequence analysis, Including the homology comparison of the primary probe with its source sequence region (i.e. <210>1) and other known genomic sequences and their complementary regions, if the homology with the non-target molecular region is greater than 85% or If there are more than 15 consecutive bases that are identical, the primary probe should generally not be used; 5. Whether the primary probe is finally selected as a probe with practical application value should be further determined by experiments. After completing the analysis of the above aspects, select and synthesize the following two probes:

探针1(probe1)，属于第一类探针，与<210>1的基因片段完全同源或互补(41Nt)：Probe 1 (probe1), which belongs to the first type of probe, is completely homologous or complementary to the <210>1 gene segment (41Nt):

5’-TGACTCTTGACTCTGCCGGGGTCTGGCTTTTTCCAGGGCTT-3’(<210>8)5'-TGACTCTTGACTCTGCCGGGGTCTGGCTTTTTCCAGGGCTT-3'(<210>8)

探针2(probe2)，属于第二类探针，相当于<210>1的基因片段或其互补片段的替换突变序列(41Nt)：Probe 2 (probe2), belonging to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the <210>1 gene fragment or its complementary fragment:

5’-TGACTCTTGACTCTGCCGGGATCTGGCTTTTTCCAGGGCTT-3’(<210>9)5'-TGACTCTTGACTCTGCCGGGATCTGGCTTTTTCCAGGGCTT-3'(<210>9)

与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文献：DNA PROBES G.H.Keller；M.M.Manak；Stockton Press，1989(USA)以及更常用的分子克隆实验手册书籍如《分子克隆实验指南》(1998年第二版)[美]萨姆布鲁克等著，科学出版社。For other unlisted common reagents and their preparation methods related to the following specific experimental steps, please refer to the literature: DNA PROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (Second Edition in 1998) [US] Sam Brook et al., Science Press.

样品制备：1，从新鲜或冰冻组织中提取DNASample preparation: 1. Extract DNA from fresh or frozen tissue

步骤：1)将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液(PBS)的平皿中。用剪刀或手术刀将组织切成小块。操作中应保持组织湿润。2)以1000g离心切碎组织10分钟。3)用冷匀浆缓冲液(0.25mol/L蔗糖；25mmol/LTris-HCl，pH7.5；25mmol/LnaCl；25mmol/L MgCl₂)悬浮沉淀(大约10ml/g)。4)在4℃用电动匀浆器以全速匀浆组织悬液，直至组织被完全破碎。5)1000g离心10分钟。6)用重悬细胞沉淀(每0.1g最初组织样品加1-5ml)，再以1000g离心10分钟。7)用裂解缓冲液重悬沉淀(每0.1g最初组织样品加1ml)，然后接以下的苯酚抽提法。2，DNA的苯酚抽提法Steps: 1) Put fresh or freshly thawed normal liver tissue into a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. The tissue should be kept moist during operation. 2) Mince the tissue by centrifugation at 1000 g for 10 minutes. 3) Suspend the pellet (approximately 10ml/g) with cold homogenization buffer (0.25mol/L sucrose; 25mmol/L Tris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl₂ ). 4) Homogenize the tissue suspension at 4°C with an electric homogenizer at full speed until the tissue is completely broken. 5) Centrifuge at 1000 g for 10 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the initial tissue sample), and then centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), and then follow the following phenol extraction method. 2. Phenol extraction of DNA

步骤：1)用1-10ml冷PBS洗细胞，1000g离心10分钟。2)用冷细胞裂解液重悬浮沉淀的细胞(1×10⁸细胞/ml)最少应用100ul裂解缓冲液。3)加SDS至终浓度为1％，如果在重悬细胞之前将SDS直接加入到细胞沉淀中，细胞可能会形成大的团块而难以破碎，并降低的总产率。这一点在抽提＞10⁷细胞时特别严重。4)加蛋白酶K至终浓度200ug/ml。5)50℃保温反应1小时或在37℃轻轻振摇过夜。6)用等体积苯酚∶氯仿∶异戊醇(25∶24∶1)抽提，在小离心机管中离心10分钟。两相应清楚分离，否则重新进行离心。7)将水相转移至新管。8)用等体积氯仿∶异戊醇(24∶1)抽提，离心10分钟。9)将含DNA的水相转移至新管。然后进行DNA的纯化和乙醇沉淀。3，DNA的纯化和乙醇沉淀Steps: 1) Wash the cells with 1-10ml of cold PBS, and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells (1×10⁸ cells/ml) with cold cell lysate and apply at least 100ul of lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break and reduce the overall yield. This is especially serious when extracting >10⁷ cells. 4) Add proteinase K to a final concentration of 200ug/ml. 5) Incubate at 50°C for 1 hour or gently shake overnight at 37°C. 6) Extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge in a small centrifuge tube for 10 minutes. The two phases were clearly separated, otherwise the centrifugation was repeated. 7) Transfer the aqueous phase to a new tube. 8) Extract with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. DNA purification and ethanol precipitation were then performed. 3. DNA Purification and Ethanol Precipitation

步骤：1)将1/10体积2mol/L醋酸钠和2倍体积冷100％乙醇加到DNA溶液中，混匀。在-20℃放置1小时或至过夜。2)离心10分钟。3)小心吸出或倒出乙醇。4)用70％冷乙醇500ul洗涤沉淀，离心5分钟。5)小心吸出或倒出乙醇。用500ul冷乙醇洗涤沉淀，离心5分钟。6)小心吸出或倒出乙醇，然后在吸水纸上倒置使残余乙醇流尽。空气干燥10-15分钟，以使表面乙醇挥发。注意不要使沉淀完全干燥，否则较难重新溶解。7)以小体积TE或水重悬DNA沉淀。低速涡旋振荡或用滴管吹吸，同时逐渐增加TE，混合至DNA充分溶解，每1-5×10⁶细胞所提取的大约加1ul。Steps: 1) Add 1/10 volume of 2mol/L sodium acetate and 2 times volume of cold 100% ethanol to the DNA solution, and mix well. Place at -20°C for 1 hour or up to overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour off the ethanol. 4) Wash the precipitate with 500 ul of 70% cold ethanol, and centrifuge for 5 minutes. 5) Carefully aspirate or pour off the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully suck out or pour off the ethanol, then invert it on absorbent paper to drain the remaining ethanol. Air dry for 10-15 minutes to allow surface ethanol to evaporate. Be careful not to dry the pellet completely as it will be difficult to redissolve. 7) Resuspend the DNA pellet with a small volume of TE or water. Vortex at a low speed or blow with a dropper, while gradually increasing TE, mix until the DNA is fully dissolved, add about 1ul for every 1-5×10⁶ cells extracted.

以下第8-13步骤仅用于必须除去污染时，否则可直接进行第14步骤。8)将RNA酶A加到DNA溶液中，终浓度为100ug/ml，37℃保温30分钟。9)加入SDS和蛋白酶K，终浓度分别为0.5％和100ug/ml。37℃保温30分钟。10)用等体积的苯酚∶氯仿∶异戊醇(25∶24∶1)抽提反应液，离心10分钟。11)小心移出水相，用等体积的氯仿∶异戊醇(24∶1)重新抽提，离心10分钟。12)小心移出水相，加1/10体积2mol/L醋酸钠和2.5体积冷乙醇，混匀置-20℃1小时。13)用70％乙醇及100％乙醇洗涤沉淀，空气干燥，重悬核酸，过程同第3-6步骤。14)测定A₂₆₀和A₂₈₀以检测DNA的纯度及产率。15)分装后存放于-20℃。样膜的制备：The following steps 8-13 are only used when the pollution must be removed, otherwise step 14 can be performed directly. 8) Add RNase A to the DNA solution with a final concentration of 100 ug/ml, and incubate at 37° C. for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug/ml respectively. Incubate at 37°C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase, re-extract with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol/L sodium acetate and 2.5 volumes of cold ethanol, mix well and place at -20°C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid, the process is the same as steps 3-6. 14) Measure A₂₆₀ and A₂₈₀ to detect the purity and yield of DNA. 15) After aliquoting, store at -20°C. Preparation of sample membrane:

1)取4×2张适当大小的硝酸纤维素膜(NC膜)，用铅笔在其上轻轻标出点样位置及样号，每一探针需两张NC膜，以便在后面的实验步骤中分别用高强度条件和强度条件洗膜。1) Take 4×2 nitrocellulose membranes (NC membranes) of appropriate size, and lightly mark the sampling position and sample number on them with a pencil. Each probe needs two NC membranes for later experiments In the step, wash the membrane with high-intensity conditions and intensity conditions respectively.

2)吸取及对照各15微升，点于样膜上，在室温中晾干。2) Pipette 15 microliters each of control and control, spot on sample membrane, and dry at room temperature.

3)置于浸润有0.1mol/LNaOH，1.5mol/LNaCl的滤纸上5分钟(两次)，晾干置于浸润有0.5mol/L Tris-HCl(pH7.0)，3mol/LNaCl的滤纸上5分钟(两次)，晾干。3) Place on filter paper soaked with 0.1mol/LNaOH, 1.5mol/LNaCl for 5 minutes (twice), dry and place on filter paper soaked with 0.5mol/L Tris-HCl (pH7.0), 3mol/LNaCl 5 minutes (twice), let dry.

4)夹于干净滤纸中，以铝箔包好，60-80℃真空干燥2小时。4) Put it in clean filter paper, wrap it with aluminum foil, and dry it under vacuum at 60-80°C for 2 hours.

探针的标记labeling of the probe

1)3μlProbe(0.1OD/10μl)，加入2μlKinase缓冲液，8-10uCiγ-³²P-dATP+2UKinase，以补加至终体积20μl。1) 3 μl Probe (0.1OD/10 μl), add 2 μl Kinase buffer, 8-10 uCi γ-³² P-dATP+2UKinase, to make up to a final volume of 20 μl.

2)37℃保温2小时。2) Incubate at 37°C for 2 hours.

3)加1/5体积的溴酚蓝指示剂(BPB)。3) Add 1/5 volume of bromophenol blue indicator (BPB).

4)过Sephadex G-50柱。4) Pass through Sephadex G-50 column.

5)至有³²P-Probe洗出前开始收集第一峰(可用Monitor监测)。5) Start to collect the first peak before³² P-Probe elutes (can be monitored by Monitor).

6)5滴/管，收集10-15管。6) 5 drops/tube, collect 10-15 tubes.

7)用液体闪烁仪监测同位素量7) Monitor the amount of isotopes with a liquid scintillation instrument

8)合并第一峰的收集液后即为所需制备的³²P-Probe(第二峰为游离γ-³²P-dATP)。8) The³² P-Probe (the second peak is free γ-³² P-dATP) is prepared after combining the collected solution of the first peak.

预杂交prehybridization

将样膜置于塑料袋中，加入3-10mg预杂交液(10×Denhardt’s；6×SSC，0.1mg/mlCT DNA(小牛胸腺DNA)。)，封好袋口后，68℃水浴摇2小时。Put the sample membrane in a plastic bag, add 3-10mg pre-hybridization solution (10×Denhardt’s; 6×SSC, 0.1mg/ml CT DNA (calf thymus DNA).), seal the bag, shake in a water bath at 68°C for 2 Hour.

杂交hybridize

将塑料袋剪去一角，加入制备好的探针，封好袋口后，42℃水浴摇过夜。Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a water bath at 42°C overnight.

洗膜：高强度洗膜：Membrane Washing: High Strength Membrane Washing:

1)取出已杂交好的样膜。1) Take out the hybridized sample membrane.

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

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

4)0.1×SSC，0.1％SDS中，55℃洗30分钟(2次)，室温晾干。低强度洗膜：4) Wash in 0.1×SSC, 0.1% SDS, 55°C for 30 minutes (twice), and dry at room temperature. Low intensity wash:

1)取出已杂交好的样膜。1) Take out the hybridized sample membrane.

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

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

4)0.1×SSC，0.1％SDS中，40℃洗15分钟(2次)，室温晾干。4) Wash in 0.1×SSC, 0.1% SDS, 40°C for 15 minutes (twice), and dry at room temperature.

X-光自显影：X-ray autoradiography:

-70℃，X-光自显影(压片时间根据杂交斑放射性强弱而定)。-70°C, X-ray autoradiography (the pressing time depends on the radioactivity of the hybridization spots).

实验结果：Experimental results:

采用低强度洗膜条件所进行的杂交实验，以上两个探针杂交斑放射性强弱没有明显区别；而采用高强度洗膜条件所进行的杂交实验，探针1的杂交斑放射性强度明显强于另一个探针杂交斑的放射性强度。因而可用探针1定性和定量地分析本发明的多核苷酸在不同组织中的存在和差异表达。In the hybridization experiment conducted under low-intensity washing conditions, there was no significant difference in the radioactivity of the hybridization spots of the above two probes; while in the hybridization experiment performed under high-intensity washing conditions, the radioactivity of the hybridization spots of probe 1 was significantly stronger than that of The radioactive intensity of another probe hybridizing spot. Probe 1 can thus be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.

                      序列表<110>复旦大学Sequence Listing <110> Fudan University

上海博德基因开发有限公司<120>一种多肽——果糖-1，6-二磷酸酶-16.61<130>4676b09<160>9<170>Patent In version 3.1<210>1<211>1231<212>DNA<213>human<220><221>CDS<222>(55)..(510)<223><400>1gacaggcaga gtgacaggga tggacggggc tgccacggga cgcccctgct cctt atg       57Shanghai Broad Gene Development Co., Ltd.<120>A Polypeptide-Fructose-1,6-bisphosphatase-16.61<130>4676b09<160>9<170>Patent In version 3.1<210>1<211>1231< 212>DNA<213>human<220><221>CDS<222>(55)..(510)<223><400>1gacaggcaga gtgacaggga tggacggggc tgccacggga cgcccctgct cctt atg 57

                                                        MetMet

                                                        1act ctt gac tct gcc ggg gtc tgg ctt ttt cca ggg ctt tgc aag cag      105Thr Leu Asp Ser Ala Gly Val Trp Leu Phe Pro Gly Leu Cys Lys Gln1ACT CTT GAC TCT GCC GGG GTC TGG CTT TTT CCA GGG CTT TGC AAG CAG 105thr Leu Aser Ala Gly Val Trp Leu Pro Gly Leu Cys Lys Lys Lys Gln Gln Gln

        5                   10                  15cag cag agg cta ctc tca ctc ttg tcc ccg tgc ccc tgc gga tgg cag      153Gln Gln Arg Leu Leu Ser Leu Leu Ser Pro Cys Pro Cys Gly Trp Gln5 10 15CAG CAG AGG CTA CTC TCA CTC TCC CCG TGC CCC CCC GGC GGG CAG 153GLN GLN ARG Leu Leu Leu Seru Cys Pro Cys Gly TRP Gln

    20                  25                  30tcg tgg gga ggg acc cca gcc agt gct tct ctg aga tgt ctg tgt gaa      201Ser Trp Gly Gly Thr Pro Ala Ser Ala Ser Leu Arg Cys Leu Cys Glu20 25 30TCG TGG GGG ACC CCA GCC AGT GCT GCT CTG AGA TGT CTG TGT GAA 201SER THR PRO ALA Serg Cys Leu Cys Glu

35                  40                  45ctg agg gaa gtc ggc tct gtc ctg ccc cag gcc agc act gac cac tcc      249Leu Arg Glu Val Gly Ser Val Leu Pro Gln Ala Ser Thr Asp His Ser50                  55                  60                  65tgg gcc ttg ctt cca aat gtc tcc ccc agt cca ctg cct aac aaa aag      297Trp Ala Leu Leu Pro Asn Val Ser Pro Ser Pro Leu Pro Asn Lys Lys35                  40                  45ctg agg gaa gtc ggc tct gtc ctg ccc cag gcc agc act gac cac tcc      249Leu Arg Glu Val Gly Ser Val Leu Pro Gln Ala Ser Thr Asp His Ser50                  55                  60                  65tgg gcc ttg ctt cca aat gtc tcc ccc agt cca ctg cct aac aaa aag 297Trp Ala Leu Leu Pro Asn Val Ser Pro Ser Pro Leu Pro Asn Lys Lys

            70                  75                  80ctc ctc tgt aat cca gct cca cag tgg aag ccc agt gga tcc tct ggc      345Leu Leu Cys Asn Pro Ala Pro Gln Trp Lys Pro Ser Gly Ser Ser Gly70 75 80CTC CTC TCA GCA GCT CCA CAG TGG CCC AGT GGA TCC TCT GGC 345leu Leu Cys Pro Ala Pro Gln Trp Lys Gly Ser Gly Gly Gly GLY

        85                  90                  95tgg gag gtg act ctc cct ggg cct ctg gta ttt ctg tgt agt tct gac      393Trp Glu Val Thr Leu Pro Gly Pro Leu Val Phe Leu Cys Ser Ser Asp85 90 95TGG GAG GAG GAG ACT CTC CCT GGG CCT CTG GTA TTT CTG TGT TCT GAC 393TRP Glu Val Thr Leu GLY PHE Leu Val Phe Leu Cyser Aser ASP

    100                 105                 110cag agc ctt tgt ttc agg ttg tct ttt cag gga cgc ttg tac agc aga      441Gln Ser Leu Cys Phe Arg Leu Ser Phe Gln Gly Arg Leu Tyr Ser Arg100 105 110CAG AGC CTT TGT TTC AGG TTG TCT TTT CAG GGA CGC TAC AGC AGC AGA 441GLN Ser Leu Cys PHE AR PHE GLN GLN GLN Leu Ty Yu Tyyr Seru -ARR -Le's Leeu's Tyr that 1 -4y Pyryr that

115                 120                 125cag cct tgg aag aaa gag aga gag tct cac tca gga gct cag gac agg      489Gln Pro Trp Lys Lys Glu Arg Glu Ser His Ser Gly Ala Gln Asp Arg130                 135                 140                 145tat gct tac tgc ctg tta taa aagattcggg ttctctaaca acagggtcat         540Tyr Ala Tyr Cys Leu Leu115                 120                 125cag cct tgg aag aaa gag aga gag tct cac tca gga gct cag gac agg      489Gln Pro Trp Lys Lys Glu Arg Glu Ser His Ser Gly Ala Gln Asp Arg130                 135                 140                 145tat gct tac tgc ctg tta taa aagattcggg ttctctaaca acagggtcat         540Tyr Ala Tyr Cys Leu Leu

            150ctcctgcaat gcacaccgct gcttgcaggg gtacctcacc ctcttcacat ccccccacgg    600gagctggggc tcaggagcca tggtgcacgg acatgctggc cctgactctg cactactggg    660gggtgataaa gacctttgta tctgacccag gagtctcgtg tcctctgcct gcattcatga    720aactgtgggt ggctacctcg ttcattcgca aggacctgtg aaaggaaaat aaatcttggg    780accgcaaagt cactaagcca aagggaaaag tcacactggg aactgtcagg cgagcctgct    840cccattttat tcccaaataa gacagataca gagattaaaa aaacaaaaaa gccacacacc    900tccctcacaa tttgtgcaca aggaaattcc ccgtggggct caagatcttt accctagagc    960agttgttggg atttcaccct ggcaatgtca gtggacagct taccttcaca ggtgcagatg   1020aaggacagaa ctcaaagcca tccctctgct cccctgagac aaacgcatat ctgactgctt   1080cctcggccct gttgtctcac tgagccagac taaggcacac gtggcccttc ccctaccccc   1140tctcacatgt ggattgtgta ttcagtgaaa ggctgatcag agactcatag aatgcaaaaa   1200aaaaaaaaca tgtcggccgc ctcggcctat g                                  1231<210>2<211>151<212>PRT<213>human<400>2Met Thr Leu Asp Ser Ala Gly Val Trp Leu Phe Pro Gly Leu Cys Lys1               5                   10                  15Gln Gln Gln Arg Leu Leu Ser Leu Leu Ser Pro Cys Pro Cys Gly Trp150ctcctgcaat gcacaccgct gcttgcaggg gtacctcacc ctcttcacat ccccccacgg    600gagctggggc tcaggagcca tggtgcacgg acatgctggc cctgactctg cactactggg    660gggtgataaa gacctttgta tctgacccag gagtctcgtg tcctctgcct gcattcatga    720aactgtgggt ggctacctcg ttcattcgca aggacctgtg aaaggaaaat aaatcttggg    780accgcaaagt cactaagcca aagggaaaag tcacactggg aactgtcagg cgagcctgct    840cccattttat tcccaaataa gacagataca gagattaaaa aaacaaaaaa gccacacacc    900tccctcacaa tttgtgcaca aggaaattcc ccgtggggct caagatcttt accctagagc    960agttgttggg atttcaccct ggcaatgtca gtggacagct taccttcaca ggtgcagatg   1020aaggacagaa ctcaaagcca tccctctgct cccctgagac aaacgcatat ctgactgctt   1080cctcggccct gttgtctcac tgagccagac taaggcacac gtggcccttc ccctaccccc   1140tctcacatgt ggattgtgta ttcagtgaaa ggctgatcag agactcatag aatgcaaaaa   1200aaaaaaaaca tgtcggccgc ctcggcctat g                                  1231<210>2<211>151<212>PRT<213>human<400>2Met Thr Leu Asp Ser Ala Gly Val Trp Leu Phe Pro Gly Leu Cys Lys1 5 5 10 15Gln Gln Gln Arg Leu Leu Ser Leu Leu Ser Pro Cys Pro Cys Gly Trp

        20                  25                  30Gln Ser Trp Gly Gly Thr Pro Ala Ser Ala Ser Leu Arg Cys Leu Cys20 25 25 30Gln Ser Trp Gly Gly Thr Pro Ala Ser Ala Ser Leu Arg Cys Leu Cys

    35                  40                  45Glu Leu Arg Glu Val Gly Ser Val Leu Pro Gln Ala Ser Thr Asp His35 40 45Glu Leu Arg Glu Val Gly Ser Val Leu Pro Gln Ala Ser Thr Asp His

50                  55                  60Ser Trp Ala Leu Leu Pro Asn Val Ser Pro Ser Pro Leu Pro Asn Lys65                  70                  75                  80Lys Leu Leu Cys Asn Pro Ala Pro Gln Trp Lys Pro Ser Gly Ser Ser50 55 60SER TRP Ala Leu PRO Asn Val Ser Pro Seru Pro ASN LYS65 70 80LYS Leu Leu CySn Pro Ala Gln TRP LYS PRLS GLY Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Serial Serr

            85                  90                  95Gly Trp Glu Val Thr Leu Pro Gly Pro Leu Val Phe Leu Cys Ser Ser85 90 95Gly Trp Glu Val Thr Leu Pro Gly Pro Leu Val Phe Leu Cys Ser Ser

        100                 105                 110Asp Gln Ser Leu Cys Phe Arg Leu Ser Phe Gln Gly Arg Leu Tyr Ser100 105 110Asp Gln Ser Leu Cys Phe Arg Leu Ser Phe Gln Gly Arg Leu Tyr Ser

    115                 120                 125Arg Gln Pro Trp Lys Lys Glu Arg Glu Ser His Ser Gly Ala Gln Asp115 120 125Arg Gln Pro Trp Lys Lys Glu Arg Glu Ser His Ser Gly Ala Gln Asp

130                 135                 140Arg Tyr Ala Tyr Cys Leu Leu145                 150<210>3<211>24<212>DNA<213>human<400>3gacaggcaga gtgacaggga tgga                                           24<210>4<211>24<212>DNA<213>human<400>4cataggccga ggcggccgac atgt                                           24<210>5<211>33<212>DNA<213>human<400>5ccccatatga tgactcttga ctctgccggg gtc                                  33<210>6<211>33<212>DNA<213>human<400>6cccgaattct tataacaggc agtaagcata cct                                  33<210>7<211>15<212>PRT<213>human<400>7Met Thr Leu Asp Ser Ala Gly Val Trp Leu Phe Pro Gly Leu Cys1               5                   10                  15<210>8<211>41<212>DNA<213>human<400>8tgactcttga ctctgccggg gtctggcttt ttccagggct t                         41<210>9<211>41<212>DNA<213>human<400>9tgactcttga ctctgccggg atctggcttt ttccagggct t                         41130                 135                 140Arg Tyr Ala Tyr Cys Leu Leu145                 150<210>3<211>24<212>DNA<213>human<400>3gacaggcaga gtgacaggga tgga                                           24<210>4<211>24<212>DNA<213>human< 400>4cataggccga ggcggccgac atgt                                           24<210>5<211>33<212>DNA<213>human<400>5ccccatatga tgactcttga ctctgccggg gtc                                  33<210>6<211>33<212>DNA<213>human<400>6cccgaattct TATAACAGC AGTAAGCATA CCT 33 <210> 7 <211> 15 <212> PRT <213> Human <400> 7MET THR Leu ASP Ser Ala Gly Val Trp Leu Gly Leu Cys1 5 10 15 <211> 41 <211> 41 <210> 41 212> DNA <213> Human <400> 8TGACTCTGA CTCTGCCCGGGGGGGGGGGCTTTTTTTTTCCAGGGCT T 41 <210> <211> 41 <212> DNA <213> Human <400> 9TGACTGCCCCGGGGGGGGGGGGGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Claims (18)

1, a kind of isolated polypeptide-fructose-1-16.61 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.

2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in＜210〉2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉2.

4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding have＜210〉2 shown in the multinuclear glycosides of the polypeptide of aminoacid sequence or its fragment, analogue, derivative

Acid;

(b) with polynucleotide (a) complementary polynucleotide; Or

(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.

6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 55-510 position sequence or＜210〉1 in the sequence of 1-1231 position.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:

(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or

(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.

9, a kind of preparation method with the active polypeptide of fructose-1-16.61 is characterized in that described method comprises:

(a) expressing under fructose-1-16.61 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of fructose-1-16.61.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with fructose-1-16.61 specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses fructose-1-16.61.

12, compound as claimed in claim 11 is characterized in that it is＜polynucleotide sequence or its segmental antisense sequences shown in 210〉1.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate fructose-1-16.61 in vivo, the method for external activity.

14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen the stand-in of fructose-1-16.61, agonist, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

17, as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with fructose-1-16.61 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.