CN1187754A

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Info

Publication number: CN1187754A
Application number: CN96194720A
Authority: CN
Inventors: A·雅因
Original assignee: Yeda Research and Development Co Ltd
Current assignee: Yeda Research and Development Co Ltd
Priority date: 1995-06-12
Filing date: 1996-06-12
Publication date: 1998-07-15

Abstract

The present invention relates to a high affinity ligand for fibroblast growth factor receptor 3(FGFR3), fibroblast growth factor 9(FGF9), which specifically binds to and activates FGFR 3. The invention also relates to methods for detecting FGFR3 with FGF9, and pharmaceutical compositions for modulating FGFR3 activity, the compositions comprising as an active ingredient FGF9, antagonists thereof, or FGF binding agents that neutralize natural circulating FGF9, to novel recombinant murine and chicken FGF9, to expression vectors comprising these recombinant FGF9, and to transgenic animals transformed with said expression vectors.

Description

Translated fromChinese

FGFR3的特异性配体FGF9FGF9, a specific ligand of FGFR3

发明领域field of invention

本发明涉及成纤维细胞生长因子9(FGF9)，一种成纤维细胞生长因子受体3(FGFR3)的新的高亲和性配体、用所述配体检测FGFR3的方法和用于调节FGFR3活性的包含FGF9，它的拮抗剂或FGF9结合剂的药学组合物。The present invention relates to fibroblast growth factor 9 (FGF9), a novel high-affinity ligand for fibroblast growth factor receptor 3 (FGFR3), methods for detecting FGFR3 using said ligand, and methods for modulating FGFR3 Active pharmaceutical compositions comprising FGF9, its antagonists or FGF9 binding agents.

成纤维细胞生长因子(FGF)包含至少有9种涉及各种生物过程包括形态发生、血管生成和组织重建的多功能多肽的家族。它们可刺激间充质至上皮和神经外胚层起源细胞的繁殖。FGF在结构上相似，但在其靶特异性和空间及时间的表达格局上不同。已对编码跨膜蛋白质酪氨酸激酶的四种FGF受体(FGFR)基因在哺乳动物中进行了克隆和鉴定，并且有在鸟类、非洲爪蟾属和果蝇属中它们的同系物的描述(Givol和Yayon，FASEB J.，6：33623369(1992))。然而功能受体蛋白质的实际数量更多，因为不同的RNA剪接和多个聚腺苷酸化位点将产生大量细胞结合的或分泌形式的变异体。除了这些高亲和性受体外，FGF可与低亲和力、高数量结合位点的硫酸乙酰肝素蛋白聚糖(HSPG)牢固结合。这些硫酸乙酰肝素调节FGF-受体结合和生物活性，并作为FGF、FGFR和适当的HSPG的功能性三联复合物形成时的一个专性内在组分。Fibroblast growth factors (FGF) comprise a family of at least nine multifunctional polypeptides involved in various biological processes including morphogenesis, angiogenesis and tissue remodeling. They stimulate the proliferation of cells of mesenchymal to epithelial and neuroectodermal origin. FGFs are structurally similar but differ in their target specificity and spatial and temporal expression patterns. Four FGF receptor (FGFR) genes encoding transmembrane protein tyrosine kinases have been cloned and characterized in mammals, and there are reports of their homologues in birds, Xenopus and Drosophila described (Givol and Yayon, FASEB J., 6:33623369 (1992)). However, the actual number of functional receptor proteins is higher, since different RNA splicing and multiple polyadenylation sites will generate a large number of variants in cell-associated or secreted forms. In addition to these high-affinity receptors, FGF binds strongly to the low-affinity, high-number binding-site heparan sulfate proteoglycan (HSPG). These heparan sulfates modulate FGF-receptor binding and biological activity and act as an obligate intrinsic component in the formation of a functional tripartite complex of FGF, FGFR and the appropriate HSPG.

由于配体和受体的变异体很多，关于FGF功能的一个主要问题是它们的配体-受体特异性。FGFR1和FGFR2与酸性FGF/FGF1及碱性FGF/FGF2的结合有相似亲和力(Dionne等人，EMBO J.，9：2685-2692(1990))。实际上所有如此测试的FGFR与FGF1和FGF4(hst/kfgf)的结合有中度至高度亲和力，这证明FGF系统有明显的丰余性。与FGFR1和2相反，发现FGFR3只与FGF1和FGF4以中度亲和力结合(Orntiz和Leder，J.Biol.Chem.，267：16305-16311(1992)；Chellaiah等人，J.Biol.Chem.，269(15)：11620-11622(1994))。现在还没有鉴定出这个受体的任一剪接形式的特异性配体。Due to the large number of ligand and receptor variants, a major question regarding FGF function is their ligand-receptor specificity. FGFR1 and FGFR2 bind with similar affinity to acidic FGF/FGF1 and basic FGF/FGF2 (Dionne et al., EMBO J., 9:2685-2692 (1990)). Virtually all FGFRs so tested bound FGF1 and FGF4 (hst/kfgf) with moderate to high affinity, demonstrating the apparent redundancy of the FGF system. In contrast to FGFR1 and 2, FGFR3 was found to only bind with moderate affinity to FGF1 and FGF4 (Orntiz and Leder, J. Biol. Chem., 267: 16305-16311 (1992); Chellaiah et al., J. Biol. Chem., 269(15):11620-11622 (1994)). No specific ligands have been identified for either splice form of this receptor.

最近，FGFR3的突变导致了软骨发育不全这种最常见的遗传侏儒症。经对软骨发育不全患者的FGFR3测序，发现在受体的跨膜区域有突变。More recently, mutations in FGFR3 lead to achondroplasia, the most common genetic dwarfism. Sequencing of FGFR3 in patients with achondroplasia revealed mutations in the transmembrane region of the receptor.

出于研究这种疾病和开发其可能的治疗药物的要求，涉及软骨发育不全的受体FGFR3的研究就需要有一种针对这种受体的、基本上不与其它三种FGFR结合的特异性配体。In order to study this disease and develop its possible therapeutic drugs, the research involving the receptor FGFR3 of achondroplasia requires a specific ligand for this receptor that does not substantially bind to the other three FGFRs. body.

通过同源性研究发现一种从人神经胶质瘤细胞系的培养物上清液纯化获得的肝素结合的神经胶质激活因子，它是FGF家族中的第九种，因此称为FGF9。发现人的FGF9可编码208个氨基酸的蛋白质，它有独特的刺激神经胶质细胞、PC-12细胞和BALB/C 3T3成纤维细胞增殖的生物活性光谱，但对内皮细胞无促细胞分裂作用(Miyamote等人，Mol.Cell.Biol.，13(7)：4251-4259(1993)；Naro等人，J.Bio1.Chem.，267：16305-16311(1993))。A heparin-binding glial activating factor purified from the culture supernatant of human glioma cell lines was found through homology research, which is the ninth member of the FGF family, so it is called FGF9. It was found that human FGF9 can encode a protein of 208 amino acids, which has a unique biological activity spectrum that stimulates the proliferation of glial cells, PC-12 cells and BALB/C 3T3 fibroblasts, but has no mitogenic effect on endothelial cells ( Miyamote et al., Mol. Cell. Biol., 13(7):4251-4259 (1993); Naro et al., J. Biol. Chem., 267:16305-16311 (1993)).

发明概要Summary of the invention

根据令人惊奇的发现，本发明涉及成纤维细胞生长因子受体3(FGFR3)的高亲和性(kD：0.25nM)配体成纤维细胞生长因子9(FGF9)，它不与FGFR1或FGFR4结合且与FGFR2仅以极低的亲和力结合。According to the surprising discovery, the present invention relates to the high affinity (kD: 0.25 nM) ligand of fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor 9 (FGF9), which does not bind to FGFR1 or FGFR4 Binds and only with very low affinity to FGFR2.

因此，本发明首先提供FGFR3特异性配体成纤维细胞生长因子9(FGF9)。这个特异性FGFR3配体用于检测和药物治疗。Therefore, the present invention firstly provides the FGFR3-specific ligand fibroblast growth factor 9 (FGF9). This specific FGFR3 ligand is used for detection and drug therapy.

这种新的FGFR3的特异性配体可用于检测样品或组织中的FGFR3，方法包括：This new FGFR3-specific ligand can be used to detect FGFR3 in samples or tissues, and the methods include:

(i)使FGF9与样品或组织接触，使得形成受体-配体对，和(i) contacting FGF9 with a sample or tissue such that a receptor-ligand pair is formed, and

(ii)检测FGFR3-FGF9对的存在，阳性的检测结果表示样品或组织中有FGFR3存在。(ii) Detecting the existence of FGFR3-FGF9 pair, a positive detection result indicates the presence of FGFR3 in the sample or tissue.

样品可以是体液如血液样品，其中有可溶的FGFR3，组织可以是从患者处例如通过软骨活检获得的组织，或者，也可以是个体的体内组织，在这种情况下检测在体内进行。The sample may be a bodily fluid such as a blood sample, in which FGFR3 is soluble, the tissue may be tissue obtained from a patient, eg, by cartilage biopsy, or it may be tissue in the individual's body, in which case the detection is performed in vivo.

检测的进行是例如用合适的可检测标记标记FGF9，然后测定任何标记是否结合到样品中的蛋白质上或结合到组织中的细胞表面上来测定FGFR3的存在。另一种检测方法可以通过用标记过的针对FGF9的抗体来进行，它可识别与FGFR3结合的FGF9。Detection is performed, for example, by labeling FGF9 with a suitable detectable label and then determining whether any label binds to proteins in the sample or to the surface of cells in the tissue to determine the presence of FGFR3. Another detection method can be performed by using a labeled antibody against FGF9, which recognizes FGF9 bound to FGFR3.

根据本发明，发现FGF9是肝素依赖的FGFR3配体。因此，根据用FGF9配体来检测FGFR3的方法，肝素最好存在于检测基质中。According to the present invention, it was found that FGF9 is a heparin-dependent FGFR3 ligand. Therefore, according to the method of detecting FGFR3 with FGF9 ligand, heparin is preferably present in the detection matrix.

根据本发明，还发现FGF9不仅与FGFR3特异性结合，也可特异性激活该受体而不激活FGFR1和FGFR4受体，而且，如果选择合适的浓度，它不会显著地激活FGFR2。这一发现使得可以制备药物组合物，它包含药学上可接受的载体和作为活性组分的有效治疗量的FGF9。这种药物组合物可用于刺激FGFR3的活性。According to the present invention, it is also found that FGF9 not only specifically binds to FGFR3, but also specifically activates this receptor without activating FGFR1 and FGFR4 receptors, and, if an appropriate concentration is selected, it does not significantly activate FGFR2. This discovery allows the preparation of pharmaceutical compositions comprising a pharmaceutically acceptable carrier and, as an active ingredient, a therapeutically effective amount of FGF9. Such pharmaceutical compositions are useful for stimulating the activity of FGFR3.

这一发现也使得可以制备药物组合物，它包括药学上可接受的载体和作为活性组分的FGF9拮抗剂，或一种FGF9结合剂如针对FGF9的一种抗体。This discovery also allows the preparation of pharmaceutical compositions comprising a pharmaceutically acceptable carrier and as an active ingredient a FGF9 antagonist, or a FGF9 binding agent such as an antibody against FGF9.

包含FGF9拮抗剂的药物组合物可直接弱化FGFR3的活性，包含FGF9结合剂如针对FGF9的抗体的药物组合物可中和循环的天然FGF9，因此间接弱化了FGFR3的活性。Pharmaceutical compositions comprising FGF9 antagonists can directly attenuate FGFR3 activity, and pharmaceutical compositions comprising FGF9-binding agents, such as antibodies against FGF9, can neutralize circulating native FGF9, thus indirectly attenuating FGFR3 activity.

普通的软骨和骨生长及软骨和骨损伤的修复需要FGFR3活性正调节和负调节间有一个特定和准确的平衡。不是理论上希望结合，假定活性FGFR3在软骨-骨分化的最初阶段是必需的，在分化后，是软骨-骨修复所需的。因此，可用包含刺激FGFR3活性的活性组分FGF9的药物组合物，通过如对损伤部位给药来促进软骨和骨修复。而且，FGFR3通常存在于间充质干细胞中，在其分化后消失。施用FGF9可稳定FGFR3，从而延长了它在分化前的有活性的时间。FGF9对于携带FGFR3的细胞也有趋化作用，并可促进这种携带FGFR3细胞(典型的为间充质干细胞)迁移至希望的部位，例如通过注射FGF9至柱的生长盘顶部。Normal cartilage and bone growth and repair of cartilage and bone damage require a specific and precise balance between upregulation and downregulation of FGFR3 activity. Without wishing to combine theoretically, it is postulated that active FGFR3 is required during the initial stages of cartilage-bone differentiation and, after differentiation, is required for cartilage-bone repair. Accordingly, pharmaceutical compositions comprising FGF9, an active ingredient that stimulates FGFR3 activity, can be used to promote cartilage and bone repair by, for example, administering to the site of injury. Moreover, FGFR3 is normally present in mesenchymal stem cells and disappears after their differentiation. Administration of FGF9 stabilizes FGFR3, thereby prolonging the time it is active prior to differentiation. FGF9 also has a chemotactic effect on FGFR3-carrying cells and can promote the migration of such FGFR3-carrying cells (typically mesenchymal stem cells) to a desired site, for example by injecting FGF9 onto the top of a growth plate of a column.

根据这个理论，在最初的骨和软骨细胞分化阶段后的FGFR3过度激活将导致生长停止，这可能就导致软骨发育不全。因此，包含可弱化FGFR3活性的活性组分FGF9的拮抗剂的药物组合物，或包含中和天然循环的FGF9的FGF9结合剂(如针对FGF9的抗体)的药物组合物可用于已分化组织中FGFR3受体过度活化的情况(这种情况形成骨和软骨生长停止)，这种骨和软骨生长停止将导致软骨发育不全侏儒症，或其它骨和软骨生长畸形，例如多发性遗传外生骨疣，单发性遗传外生骨疣、趾外翻畸形、滑液软骨瘤和内软骨瘤。According to this theory, overactivation of FGFR3 after the initial stages of bone and chondrocyte differentiation would lead to growth arrest, which could lead to achondroplasia. Accordingly, pharmaceutical compositions comprising antagonists of the active component FGF9 that attenuate FGFR3 activity, or pharmaceutical compositions comprising FGF9-binding agents (such as antibodies against FGF9) that neutralize naturally circulating FGF9 can be used to treat FGFR3 in differentiated tissues. Conditions of overactivation of the receptors (which lead to cessation of bone and cartilage growth) leading to achondroplasia dwarfism, or other bone and cartilage growth malformations such as multiple inherited exostoses, Solitary hereditary exostosis, hallux valgus, synovial chondroma, and endochondroma.

上述疾病可用包含FGF9的拮抗剂或可中和天然循环的FGF9的FGF9结合剂的药物组合物来治疗，其中两种组分均可弱化FGFR3的活性。The above diseases can be treated with pharmaceutical compositions comprising antagonists of FGF9 or FGF9 binding agents that neutralize naturally circulating FGF9, both of which attenuate the activity of FGFR3.

本发明也涉及一种新的重组鼠FGF9，和一种新的重组鸡FGF9以及编码这些新的重组蛋白的DNA序列。The present invention also relates to a novel recombinant mouse FGF9, a novel recombinant chicken FGF9 and DNA sequences encoding these novel recombinant proteins.

本发明还涉及一种包含FGF9序列的用强启动子(如CMV或SV40)或软骨/骨启动子(如胶原2型启动子)控制表达的表达载体。这种表达载体可形成一个过量表达FGF9的转基因哺乳动物，导致FGFR3受体的过度激活从而引起生长停止。这种动物可用作生长停止性疾病如遗传性软骨发育不全的模型。The present invention also relates to an expression vector containing FGF9 sequence controlled by a strong promoter (such as CMV or SV40) or cartilage/bone promoter (such ascollagen type 2 promoter). This expression vector produces a transgenic mammal overexpressing FGF9, leading to overactivation of the FGFR3 receptor and causing growth arrest. Such animals are useful as models for growth arrest disorders such as hereditary achondroplasia.

下面发明将参照一些无限制性的附图和实施例来描述。The following invention will be described with reference to some non-limiting figures and examples.

附图详述Detailed description of the drawings

图1-鼠FGF9的核苷酸和氨基酸序列，其中显示了FGF9：pET-3C和推导的氨基酸序列。Figure 1 - Nucleotide and amino acid sequence of murine FGF9 showing FGF9: pET-3C and deduced amino acid sequence.

图2-鸡FGF9的核苷酸和氨基酸序列，其中显示了FGF9：pET-3C和推导的氨基酸序列。Figure 2 - Nucleotide and amino acid sequence of chicken FGF9 showing FGF9: pET-3C and deduced amino acid sequence.

图3A、3B、3C显示了鼠，大鼠和人FGF9的氨基酸和核苷酸序列的比较。Figures 3A, 3B, 3C show a comparison of the amino acid and nucleotide sequences of murine, rat and human FGF9.

图4-FGF9的纯化。部分纯化的FGF9与肝素琼脂糖凝胶结合并用0.2-2M盐梯度洗脱，蛋白质含量用分光光度计(A)测定。为鉴别洗脱组分中的FCF9，将每一组分10ml用15％SDS PAGE分离，转移至硝基纤维素上，用特异性抗体(抗SP32)(B)进行免疫印迹。组分的纯度通过在15％SDS PAGE(C)中分离的每一个组分中加入5ml银染色剂来测定。Figure 4 - Purification of FGF9. Partially purified FGF9 was bound to heparin sepharose and eluted with a 0.2-2M salt gradient, and the protein content was determined with a spectrophotometer (A). To identify FCF9 in the eluted fractions, 10 ml of each fraction was separated by 15% SDS PAGE, transferred to nitrocellulose, and immunoblotted with a specific antibody (anti-SP32) (B). The purity of the fractions was determined by adding 5 ml of silver stain to each fraction separated in 15% SDS PAGE (C).

图5-FGF9的结合特异性。将纯化的FGF9固定在肝素琼脂糖凝胶珠上，测定FGF9与碱性磷酸酶连接的各种FGFR的可溶胞外区域的结合能力(A)。FGFR的量根据碱性磷酸酶活性(B)来估计。FGFR1、2、7-IIIb、3、3-IIIb和4碱性磷酸酶融合蛋白的等量的可溶胞外区域可用抗碱性磷酸酶抗体来免疫沉淀。在有或没有0.5μg/ml肝素及百倍过量的未标记的FGF9(过量未标记)时用所述的材料和方法使¹²⁵I-FGF9结合和交联。Figure 5 - Binding specificity of FGF9. Purified FGF9 was immobilized on heparin-sepharose beads, and the ability of FGF9 to bind to the soluble extracellular domain of various FGFRs linked to alkaline phosphatase was determined (A). The amount of FGFR was estimated from alkaline phosphatase activity (B). Equal amounts of soluble extracellular domains of FGFR1, 2, 7-IIIb, 3, 3-IIIb, and 4 alkaline phosphatase fusion proteins were immunoprecipitated with anti-alkaline phosphatase antibodies.¹²⁵ I-FGF9 was bound and cross-linked using the described materials and methods with or without 0.5 μg/ml heparin and a hundred-fold excess of unlabeled FGF9 (excess unlabeled).

图6-与可溶性FGFR2和FGFR3结合的FGF9的分析。在所述的材料和方法下，使浓度增加的¹²⁵I-FGF9与吸附在maxisorb板盘上的FGFR2(A)和FGFR3(B)的可溶胞外区域结合。结合结果用Scatchard分析法(插入小图)分析。Figure 6 - Analysis of FGF9 binding to soluble FGFR2 and FGFR3. Using the materials and methods described, increasing concentrations^of125I -FGF9 were bound to the soluble extracellular domains of FGFR2 (A) and FGFR3 (B) adsorbed on maxisorb plates. Binding results were analyzed by Scatchard analysis (insert panel).

图7-FGF9与表达FGFR3的CHO细胞的肝素依赖性交联。如所述40℃时在有或没有1mg/ml肝素及100倍过量的未标记过FGF9(过量未标记)下，5ng/ml¹²⁵I-FGF9与单层的FGFR3转染的K1和A745 CHO细胞培育。如用所述材料和方法进行交联和电泳分离。Figure 7 - Heparin-dependent cross-linking of FGF9 to FGFR3-expressing CHO cells. K1 and A745 CHO cells transfected with 5 ng/ml¹²⁵ I-FGF9 and a monolayer of FGFR3 with or without 1 mg/ml heparin and a 100-fold excess of unlabeled FGF9 (excess unlabeled) at 40°C as described nurture. Cross-linking and electrophoretic separation were performed as described with materials and methods.

图8-肝素和肝素组分依赖的FGF9诱导的DNA合成。使单层FGFR3转染的CHO-A745细胞处于血清饥饿状态，并和10ng/ml FGF9、指定量的肝素(A)或2mg/ml指定数目单糖单元的肝素组分(B)进行培育。Figure 8 - Heparin and heparin component dependent FGF9-induced DNA synthesis. Monolayers of FGFR3-transfected CHO-A745 cells were serum-starved and incubated with 10 ng/ml FGF9, indicated amounts of heparin (A) or 2 mg/ml heparin fractions of the indicated number of monosaccharide units (B).

图9-由胶原2型启动子控制的FGF9的质粒。Figure 9 - Plasmid of FGF9 controlled by thecollagen type 2 promoter.

发明详述Detailed description of the invention

I.材料和方法I. Materials and methods

(a)细胞(a) cell

用F12培养基附加10％胎牛血清来培育Dr.J.D.Esko(生物化学系，伯明翰大学，阿拉巴马州)提供的野生型(KI)和CHO突变型细胞系A745。用有新霉素抗性的pZL质粒中的10μgFGFR3转染CHO细胞是通过用Gene Pulcer(Bio-Rad)在960微法，250伏下进行电穿孔来实现的。用G418(0.5mg/ml)来选择单个稳定的克隆。Wild-type (KI) and CHO mutant cell line A745 provided by Dr. J.D. Esko (Department of Biochemistry, University of Birmingham, Alabama) was grown in F12 medium supplemented with 10% fetal calf serum. Transfection of CHO cells with 10 μg of FGFR3 in the neomycin-resistant pZL plasmid was achieved by electroporation with a Gene Pulcer (Bio-Rad) at 960 microfarads, 250 volts. Single stable clones were selected with G418 (0.5 mg/ml).

(b)抗体(b) Antibody

抗FGF9多克隆抗体是通过对新西兰白兔注射并在两个附加的剂量后收集血清来产生。抗FGF9抗体用MBS针对与KLH(匙孔血蓝蛋白)偶连的两个肽段(SP31：Cys-Ser-Asn-Leu-Tyr-Lys-His-Val-Gln-Thr-Gly-Arg-Arg-Tyr，SP32：Asp-His-Leu-Lys-Gly-Ile-Leu-Arg-Arg-Arg-Gln-Leu-Tyr-Cys)来制备。获得的血清在蛋白质A琼脂糖凝胶(Repligen)上进一步纯化来获得IgG组分。Anti-FGF9 polyclonal antibodies were raised by injecting New Zealand white rabbits and collecting serum after two additional doses. The anti-FGF9 antibody was directed against two peptides (SP31: Cys-Ser-Asn-Leu-Tyr-Lys-His-Val-Gln-Thr-Gly-Arg- Arg-Tyr, SP32: Asp-His-Leu-Lys-Gly-Ile-Leu-Arg-Arg-Arg-Gln-Leu-Tyr-Cys) to prepare. The serum obtained was further purified on protein A sepharose (Repligen) to obtain the IgG fraction.

(c)FGF9的放射性标记(c) Radiolabeling of FGF9

重组鼠FGF9用Na¹²⁵I(0.5mCi)通过氯胺T方法作标记，并在肝素琼脂糖凝胶柱上与游离的碘分离。比活范围是0.5-2×10⁵cpm/ng。Recombinant murine FGF9 was labeled with Na¹²⁵ I (0.5 mCi) by the chloramine T method and separated from free iodine on a heparin sepharose column. The specific activity range is 0.5-2×10⁵ cpm/ng.

实施例1：FGF9的鼠同系物的克隆和表达Example 1: Cloning and Expression of the Murine Homolog of FGF9

从12.5天的鼠胚胎中抽提出全部RNA用于基于FGF9克隆的聚合酶链反应(PCR)。用人FGF9的特异性引物(正向：GGGAATTCCATATGGCTCCCTTAGGTGAAG；反向：CGGGATCCTCAACTTTGGCTTAGAATATCC)及鼠RNA模板进行PCR。(35个循环，每个循环在94℃变性1分钟，在56℃退火2分钟，72℃延伸3分钟)。获得单一的预计大小为630bp的DNA产物，其直接用于亚克隆人pET-3C细菌表达载体(Novagene)。Total RNA was extracted from 12.5-day-old mouse embryos for polymerase chain reaction (PCR) based on FGF9 cloning. PCR was performed with specific primers for human FGF9 (forward: GGGAATTCCATATGGCTCCCTTAGGTGAAG; reverse: CGGGATCCTCAACTTTGGCTTAGAATATCC) and mouse RNA template. (35 cycles, each cycle of denaturation at 94°C for 1 minute, annealing at 56°C for 2 minutes, and extension at 72°C for 3 minutes). A single DNA product with an expected size of 630 bp was obtained, which was directly used for subcloning the human pET-3C bacterial expression vector (Novagene).

序列分析揭示预计为672bp长的转录物(图1)与人FGF9 cDNA有93％相同。FGF9：pET-3C质粒用于转化大肠杆菌的B1-21菌株。在对数生长期，转化的细菌用lmMIPTG诱导2小时，在7000RPM下离心沉淀，并用探头超声发生器(Soniprep150，MSE)在冰上超声处理3个15秒。将细菌超声破碎物离心后获得的上清液上样于肝素琼脂糖凝胶柱(Pharmacia，Upsala，Sweden)，柱用10倍柱体积的0.15MNaCl，0.05％Chaps，20mM Tris pH7.4和10倍柱体积的0.7M NaCl，0.05％Chaps，20mM Tris pH7.4充分洗涤，然后，结合的蛋白质在0.5ml的2M NaCl，0.05％Chaps，10mM Tris pH7.4组分处洗脱，用水1∶10稀释并重新上柱于预平衡的1ml肝素琼脂糖凝胶小型FPLC柱(Pharmacia，Upsala，Sweden)。在充分洗柱后，柱用连续的0.2-2M NaCl梯度洗脱，并通过280nm处的吸光度来决定蛋白质曲线。组分通过在BALB/c-3T3成纤维细胞中掺入3H-胸苷来测定其生物活性，通过Western印迹法用在兔中产生的针对FGF9特异性多肽的多克隆抗体来测定特异性。在预计分子量27kDal处获得的主要蛋白质条带可与两个不同的特异性针对FGF9肽段的抗体发生特异性反应。Sequence analysis revealed that the predicted 672 bp long transcript (Figure 1) was 93% identical to human FGF9 cDNA. The FGF9:pET-3C plasmid was used to transform the B1-21 strain of E. coli. In the logarithmic growth phase, transformed bacteria were induced with lmMIPTG for 2 hours, pelleted by centrifugation at 7000 RPM, and sonicated 3 times for 15 seconds on ice with a probe sonicator (Soniprep 150, MSE). The supernatant obtained after the bacterial sonication was centrifuged was loaded on a heparin sepharose column (Pharmacia, Upsala, Sweden) with 10 column volumes of 0.15M NaCl, 0.05% Chaps, 20mM Tris pH7.4 and 10 The column volume of 0.7M NaCl, 0.05% Chaps, 20mM Tris pH7.4 was fully washed, and then, the bound protein was eluted at the component of 0.5ml of 2M NaCl, 0.05% Chaps, 10mM Tris pH7.4, and water 1: 10 diluted and reloaded on a pre-equilibrated 1 ml Heparin Sepharose Mini FPLC column (Pharmacia, Upsala, Sweden). After washing the column thoroughly, the column was eluted with a continuous 0.2-2M NaCl gradient, and the protein profile was determined by the absorbance at 280nm. The biological activity of the components was determined by incorporation of 3H-thymidine in BALB/c-3T3 fibroblasts, and the specificity was determined by Western blotting with polyclonal antibodies raised in rabbits against the FGF9-specific polypeptide. The main protein band obtained at the estimated molecular weight of 27kDal can specifically react with two different antibodies specific for the FGF9 peptide.

用从12.5天的鼠胚胎RNA制得的cDNA进行PCR以克隆鼠FGF9(mFGF9)。鼠FGF9 cDNA与人和大鼠的FGF9分别有93％和98％的序列同源性(图3A-3C)。mFGF9的氨基酸序列与大鼠FGF9的氨基酸序列相同，与人FGF9的氨基酸序列仅在位置9处不同(其为丝氨酸，而人FGF9为天冬酰胺)。重组的鼠FGF9在大肠杆菌的B1-21菌株中表达，并从细菌裂解物中通过在肝素琼脂糖凝胶柱上纯化两个循环而获得。FGF9用1.0-1.2M NaCl从肝素琼脂糖凝胶上洗脱，并在280nm测定吸光度(图4A)。组分中FGF9的存在通过免疫印迹用针对FGF9特异性肽段的多克隆抗体来测定，这证明在预计分子量处的主要蛋白质条带是27kDal的非糖基化蛋白质(如图4B)。每一个制剂的纯度还用银染法(图4C)来进一步测定。重组鼠FGF9有生物活性，它以剂量依赖性方式刺激BALB/C 3T3成纤维细胞中的DNA的合成，最大3H-胸苷掺入值的一半为0.5ng/ml(未显示)，这与从纯化的人FGF9获得的结果相同(Nauro等人，J.Bio1.Chem.，267：16305-16311(1993))。Murine FGF9 (mFGF9) was cloned by PCR using cDNA prepared from RNA of day 12.5 mouse embryos. Murine FGF9 cDNA has 93% and 98% sequence homology with human and rat FGF9, respectively (Fig. 3A-3C). The amino acid sequence of mFGF9 is identical to that of rat FGF9 and differs from that of human FGF9 only at position 9 (which is a serine, whereas human FGF9 is an asparagine). Recombinant murine FGF9 was expressed in E. coli strain B1-21 and obtained from bacterial lysates by two cycles of purification on heparin sepharose columns. FGF9 was eluted from the heparin-agarose gel with 1.0-1.2M NaCl, and the absorbance was measured at 280 nm (Fig. 4A). The presence of FGF9 in the fractions was determined by immunoblotting with polyclonal antibodies directed against FGF9-specific peptides, which demonstrated that the major protein band at the predicted molecular weight was a non-glycosylated protein of 27 kDal (Figure 4B). The purity of each preparation was further determined by silver staining (Fig. 4C). Recombinant murine FGF9 was biologically active and stimulated DNA synthesis in BALB/C 3T3 fibroblasts in a dose-dependent manner with half the maximum 3H-thymidine incorporation at 0.5 ng/ml (not shown), which was comparable to that from The same results were obtained with purified human FGF9 (Nauro et al., J. Biol. Chem., 267:16305-16311 (1993)).

实施例2：鸡FGF9同系物的克隆和表达Example 2: Cloning and expression of chicken FGF9 homologues

鸡FGF9同系物的克隆和表达用从鸡上衍生获得的mRNA如实施例1所述的方法进行。Cloning and expression of the chicken FGF9 homologue was performed as described in Example 1 using mRNA derived from chicken.

实施例3：无细胞结合测定Example 3: Cell-free binding assay

以前已经描述了鼠FGFR1，FGFR2，角质形成细胞生长因子受体(KGFR)和碱性磷酸酶表达载体中的FGFR3的两种同工型的胞外区域(Givol D.和YayonA.，Adv.Cancer Res.160，1-41(1993)；(Lev等人，生物化学，267，15970-15977(1992))。从NIH 3T3转染细胞的条件培养基中收集FGFR-碱性磷酸酶融合蛋白并直接用于结合测定。受体蛋白质的含量通过在405nm用对硝基苯酚磷酸酯作为底物(如Lev等人，同上所描述的)对碱性磷酸酶的活性进行分光光度计检测来确定。可溶的受体结合反应混合物包括受体-AP条件培养基、放射性标记的配体和肝素或其它HSPG。结合的复合物用抗碱性磷酸酶的多克隆抗体(Zymed)和蛋白质A琼脂糖凝胶(Repligen)来免疫沉淀。所有的组分在室温下混合在总体积为250ml的结合缓冲液(DMEM，其中另加入25mMHepes，pH7.4和0.1％牛血清白蛋白)中。结合反应在室温下进行2小时。结合的配体用微离心机(-2000g)在6000rpm离心10秒来回收，并用150nM NaCl，0.1％Triton-X-100和50mM Hepes，pH7.4(HNTG)的溶液来洗三次。¹²⁵I-结合的因子通过γ-计数器直接计数管子来测定。在洗涤后，室温下将0.15mM二琥珀酰亚胺基辛二酸酯(disccinimidyl，DSS)或1mMBis(磺基琥珀酰亚胺基)辛二酸酯(BS3)加入磷酸盐缓冲液(PBS)中进行交联反应30分钟。复合物用PBS洗涤两次，并用样品缓冲液煮沸5分钟。样品在SDS聚丙烯酰胺凝胶上在还原条件下用电穿孔法分离，干燥凝胶并将其置于Kodak(Eastman Kodak Co.，Rochester，NY)X-Omat AR胶片下。The extracellular regions of murine FGFR1, FGFR2, keratinocyte growth factor receptor (KGFR) and two isoforms of FGFR3 in alkaline phosphatase expression vectors have been described previously (Givol D. and Yayon A., Adv. Cancer Res.160,1-41 (1993); (Lev et al., Biochemistry, 267,15970-15977 (1992)).From the conditioned medium of NIH 3T3 transfection cell, collect FGFR-alkaline phosphatase fusion protein and Used directly in binding assays.Amounts of receptor protein were determined by spectrophotometric detection of alkaline phosphatase activity at 405 nm using p-nitrophenol phosphate as substrate (as described by Lev et al., supra). Soluble receptor-binding reaction mixture includes receptor-AP conditioned medium, radiolabeled ligand, and heparin or other HSPG. The bound complex is treated with a polyclonal antibody against alkaline phosphatase (Zymed) and protein A sepharose Gel (Repligen) came immunoprecipitation.All components were mixed at room temperature in a total volume of 250ml of binding buffer (DMEM, wherein additionally added 25mMHepes, pH7.4 and 0.1% bovine serum albumin).The binding reaction was Carried out at room temperature for 2 hours. The bound ligand was recovered by centrifugation at 6000rpm for 10 seconds in a microcentrifuge (-2000g), and decontaminated with a solution of 150nM NaCl, 0.1% Triton-X-100 and 50mM Hepes, pH 7.4 (HNTG). Wash three times.¹²⁵ I-binding factors are determined by gamma-counter direct counting tubes. After washing, 0.15 mM disuccinimidyl suberate (disccinimidyl, DSS) or 1 mM Bis (sulfosuccinyl Imino) suberate (BS3) was added to phosphate buffered saline (PBS) for cross-linking reaction for 30 minutes. The complex was washed twice with PBS and boiled with sample buffer for 5 minutes. The sample was gelled in SDS polyacrylamide The gel was separated by electroporation under reducing conditions, and the gel was dried and mounted on Kodak (Eastman Kodak Co., Rochester, NY) X-Omat AR film.

或者，使预先过夜包被抗人胎盘碱性磷酸酶的单克隆抗体(Sigma Chemicals，Isrcal)的96孔maxisorb板(Nunk)与受体-AP融合蛋白在室温下反应2小时。在用结合缓冲液洗涤后，板在室温下与不同浓度的¹²⁵I标记的FGF9在有或没有肝素时培育2小时。在培育时间的最后，板用结合缓冲液洗涤两次，并用含1.6MNaCl的20mM，pH5.4的醋酸钠洗脱。酸抽提物在γ计数器中计数。Alternatively, 96-well maxisorb plates (Nunk) pre-coated overnight with monoclonal antibody against human placental alkaline phosphatase (Sigma Chemicals, Isrcal) were reacted with receptor-AP fusion protein for 2 hours at room temperature. After washing with binding buffer, plates were incubated with different concentrations^of125I -labeled FGF9 with or without heparin for 2 hours at room temperature. At the end of the incubation time, the plates were washed twice with binding buffer and eluted with 20 mM sodium acetate, pH 5.4, containing 1.6 M NaCl. Acid extracts were counted in a gamma counter.

为说明FGF9的受体结合性质，利用了一系列的与人胎盘碱性磷酸酶偶连的FGF受体的胞外区域。如前面所证实的，FGF受体的可溶胞外区域可成功地特异性地与配体反应，从而为分析配体-受体的特异性提供了一个很好的工具(Rimion，D.L，Prof.Clin.Bi01.Res.187,131-140(1985)，Lev等人，同上)。FGF9与可溶受体间的反应首先通过测定固定在肝素-琼脂糖凝胶上的FGF9的碱性磷酸酶活性来分析。固定在肝素-琼脂糖凝胶上的FGF9与FGFR2和FGFR3融合蛋白结合，但不与FGFR1或FGFR4结合(图5A)。仅仅FGFR2和FGFR3的IIIc同I型与FGF9结合，而这些受体的IIIb同I型没有显示出与FGF9特异性结合。FGF9与可溶受体间的反应进一步通过直接使放射性标记过的FGF9结合和交联来分析(图5B)。在0.5mg/ml肝素存在时，FGF9只与FGFR2和FGFR3结合而不和FGFR1或FGFR4结合以及不和测试的任何一种IIIb剪接同I型结合。在无肝素时，没有发现有显著的结合，这表示肝素对高亲和性FGF9-受体结合的专性作用。FGF9与FGFR2和FGFR3的共价连接的两个复合物更有可能是受体-配体复合物的单体和二聚体形式。用¹²⁵I-FGF9亲和标记的可溶的FGFR2和FGFR3蛋白质在100倍摩尔过量的未标记配体中消除，这表明这些受体的结合和标记是特异性的。To illustrate the receptor binding properties of FGF9, a series of extracellular domains of the FGF receptor coupled to human placental alkaline phosphatase were used. As previously demonstrated, the soluble extracellular domain of the FGF receptor can successfully react specifically with the ligand, thus providing a good tool for analyzing ligand-receptor specificity (Rimion, DL, Prof. . Clin. BiOl. Res. 187, 131-140 (1985), Lev et al., supra). The interaction between FGF9 and soluble receptors was first analyzed by measuring the alkaline phosphatase activity of FGF9 immobilized on heparin-agarose gel. FGF9 immobilized on heparin-agarose gel bound FGFR2 and FGFR3 fusion proteins, but not FGFR1 or FGFR4 (Fig. 5A). Only the IIIc receptors of FGFR2 and FGFR3 bind to FGF9, whereas the IIIb receptors of these receptors did not show specific binding to FGF9. The interaction between FGF9 and soluble receptors was further analyzed by directly binding and cross-linking radiolabeled FGF9 (Fig. 5B). In the presence of 0.5 mg/ml heparin, FGF9 bound only to FGFR2 and FGFR3 but not to FGFR1 or FGFR4 and not to type I with any of the IIIb splices tested. In the absence of heparin, no significant binding was found, indicating an obligatory effect of heparin on high affinity FGF9-receptor binding. The two covalently linked complexes of FGF9 with FGFR2 and FGFR3 are more likely monomeric and dimeric forms of the receptor-ligand complex. Soluble FGFR2 and FGFR3 proteins affinity-labeled^with125I -FGF9 were eliminated in a 100-fold molar excess of unlabeled ligand, suggesting that binding and labeling of these receptors is specific.

为定量描述FGF9和FGFR2及FGFR3的结合，进行放射性标记的FGF9与可溶受体的直接结合测定。FGF9与两个受体的结合是特异性的并是饱和的(图6A和6B)。通过Scatchard分析方法的分析结果(图6，插入小图)表明FGF9与FGFR2结合的解离常数为2.38nM，与FGFR3反应的解离常数为0.78nM。两个附加的试验得到非常相似的结果。在单一试验中，与FGFR2的亲和力与FGFR3相比要低约3倍。FGF9与FGFRI的结合没有显著性和特异性(未显示)。To quantify the binding of FGF9 to FGFR2 and FGFR3, direct binding assays of radiolabeled FGF9 to soluble receptors were performed. Binding of FGF9 to both receptors was specific and saturable (Figures 6A and 6B). The analysis results by Scatchard analysis method (FIG. 6, inset) showed that the dissociation constant of FGF9 binding to FGFR2 was 2.38 nM, and the dissociation constant of FGFR3 was 0.78 nM. Two additional experiments gave very similar results. In a single assay, the affinity to FGFR2 was about 3-fold lower compared to FGFR3. Binding of FGF9 to FGFRI was not significant and specific (not shown).

实施例4：FGF9与细胞表面受体的高亲和性结合和交联Example 4: High affinity binding and crosslinking of FGF9 to cell surface receptors

将24了孔盘(Nunk)中的铺满培养物预冷至4℃并用结合缓冲液洗涤两次。然后使它们在4℃与不同浓度的¹²⁵I-FGF9在有或没有肝素情况下在结合缓冲液中培育2小时。弃去结合培养基，细胞用结合缓冲液洗涤两次，用含0.5M NaCl的25mM Hepes pH7.5洗涤一次。用含1.6M NaCl的20mM，pH4.5乙酸钠洗脱结合的因子并在γ计数器中计数来测定高亲和性结合的因子。根据在100倍过量的无标记因子中的高亲和性结合获得的数值确定结合反应无特异性。为进行交联，在PBS中进行结合反应，在培育1小时后，加入DSS至最终浓度为0.15M并反应1小时以上。细胞在PBS中洗涤两次，在小体积的裂解缓冲液中刮碎并裂解，裂解缓冲液含有150mM NaCl，20mM Tris(pH8.0)，1mM MgCl₂，0.1mM ZnCl₂，0.5％NP-40，1mg抑蛋白酶肽，1mg/ml亮抑蛋白酶肽和2mM PMSF。使离心澄清化的细胞裂解物煮沸并在还原条件下在SDS聚丙烯酰胺凝胶上进行电穿孔。Confluent cultures in 24 well plates (Nunk) were precooled to 4°C and washed twice with binding buffer. They were then incubated for 2 hours at 4°C with different concentrations^of125I -FGF9 in binding buffer with or without heparin. The binding medium was discarded and the cells were washed twice with binding buffer and once with 25 mM Hepes pH 7.5 containing 0.5 M NaCl. High affinity bound factor was determined by eluting bound factor with 20 mM sodium acetate, pH 4.5 containing 1.6 M NaCl and counting in a gamma counter. Binding reactions were determined to be nonspecific based on values obtained for high affinity binding in a 100-fold excess of unlabeled factor. For cross-linking, the binding reaction was performed in PBS, and after 1 hour of incubation, DSS was added to a final concentration of 0.15M and reacted for more than 1 hour. Cells were washed twice in PBS, scraped and lysed in a small volume of lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 8.0), 1 mM MgCl₂ , 0.1 mM ZnCl₂ , 0.5% NP-40 , 1 mg aprotinin, 1 mg/ml leupeptin and 2 mM PMSF. Cell lysates clarified by centrifugation were boiled and electroporated on SDS polyacrylamide gels under reducing conditions.

如上所述，FGF9与FGFR2和FGFR3的结合严格依赖于肝素的存在。为比较FGF9与每个受体的结合对肝素的特异性需要，我们首先测定FGF9与可溶FGFR2和FGFR3的结合所需的肝素量。在交联试验中，在FGFR2或FGFR3中没有加入肝素时只发现很少的复合物。然而，随着肝素浓度的增加，发现两种受体对肝素的需求有显著的不同。As mentioned above, the binding of FGF9 to FGFR2 and FGFR3 is strictly dependent on the presence of heparin. To compare the specific requirement for heparin for FGF9 binding to each receptor, we first determined the amount of heparin required for FGF9 binding to soluble FGFR2 and FGFR3. In the cross-linking assay, only few complexes were found in FGFR2 or FGFR3 without the addition of heparin. However, as the heparin concentration increased, significant differences in heparin requirements were found for the two receptors.

与碱性磷酸酶相连的FGFR2和FGFR3的可溶胞外区域用抗碱性磷酸酶的抗体来免疫沉淀，并与5ng/ml¹²⁵I-FGF9及增加浓度的肝素培育。交联和电泳分离如实施例3所述的那样进行。FGF9与FGFR2(图5A)和FGFR3(图5B)的结合量用光密度测定法来定量。The soluble extracellular domains of FGFR2 and FGFR3 linked to alkaline phosphatase were immunoprecipitated with an antibody against alkaline phosphatase and incubated with 5 ng/ml¹²⁵ I-FGF9 and increasing concentrations of heparin. Crosslinking and electrophoretic separation were performed as described in Example 3. The amount of FGF9 bound to FGFR2 (FIG. 5A) and FGFR3 (FIG. 5B) was quantified by densitometry.

FGF9与FGFR2的结合对肝素很敏感，只要加入少达0.5ng/ml的肝素就可使结合有明显的增加，最大受体结合为约5ng/ml。然而，FGF9与FGFR3的结合需要约20倍的较高水平的游离肝素，而最大受体结合只有约100ng/ml肝素，在肝素浓度大于500ng/ml时结合稍有抑制。The combination of FGF9 and FGFR2 is very sensitive to heparin, as long as adding as little as 0.5ng/ml of heparin can significantly increase the binding, the maximum receptor binding is about 5ng/ml. However, the binding of FGF9 to FGFR3 requires about 20-fold higher levels of free heparin, while the maximum receptor binding is only about 100 ng/ml heparin, and the binding is slightly inhibited when the heparin concentration is greater than 500 ng/ml.

FGF9与FGFR2或FGFR3的结合所需肝素水平间的不同可能说明，FGFR3结合需要一个更有特异性的肝素结构，其包含所用肝素混合物的相对较小的部分。为研究促进FGF9结合所需的肝素结构，我们测定了一系列大小为4至18的单糖单元的肝素组分对于FGF9与FGFR2和FGFR3的可溶胞外区域的结合的影响。The difference between the levels of heparin required for the binding of FGF9 to FGFR2 or FGFR3 may suggest that FGFR3 binding requires a more specific heparin structure comprising a relatively small fraction of the heparin mixture used. To investigate the heparin structure required to facilitate FGF9 binding, we determined the effect of a range of heparin components ranging in size from 4 to 18 monosaccharide units on the binding of FGF9 to the soluble extracellular domains of FGFR2 and FGFR3.

为了确定体内观察到的高亲和性、依赖于肝素的FGF9与FGFR3间的反应的生理关系，在野生型(KI)和硫酸乙酰肝素缺陷的突变型(745pgs)CHO细胞(该细胞已知可低水平表达内源性FGFR(Yayon A.，等人，细胞，64：841-848(1991)))中表达全长鼠FGFR3。由于未转染的细胞没显示出可检测的放射性标记FGF9的结合，也没表示出共价交联的蛋白质，FGFR3转染的CHO-KI细胞显示出145kDal的蛋白质条带，其与受体FGF9复合物的单体相对应(图7)。如所预料的，¹²⁵I-FGF9与表达FGFR3的野生型CHO-KI细胞的结合和交联不受外源性肝素的影响。然而，在没有肝素时，没有检测到FGF9与表达FGFR3的突变型HS缺陷型CHO-745细胞有交联(图7)，这支持了FGF9高效亲和反应需要有肝素类分子的观点。当加入肝素后，745-FGF3与¹²⁵I-FGF9亲和标记是显著的，与野生型细胞无法区分，这说明肝素可支持FGF9和FGFR3间的高亲和性结合。两种细胞上的结合均是特异性的和饱和的(在1mg/ml肝素存在时)，CHO-KI细胞和CHO745细胞的kD分别为0.06和0.1nM。在FGF9与CHO 745-FGFR3转染细胞结合时获得典型的肝素剂量依赖型增长，最大特异性结合为约500ng/ml的肝素(数据未列出)。To determine the physiological relationship observed in vivo for the high-affinity, heparin-dependent response between FGF9 and FGFR3, wild-type (KI) and heparan sulfate-deficient mutant (745 pgs) CHO cells known to Low-level expression of endogenous FGFR (Yayon A., et al., Cell, 64:841-848 (1991 )) Full-length murine FGFR3 was expressed. As untransfected cells showed no detectable binding of radiolabeled FGF9 nor covalently cross-linked protein, FGFR3-transfected CHO-KI cells showed a protein band of 145 kDal, which is associated with the receptor FGF9 The monomers of the complexes correspond (Fig. 7). As expected, the binding and cross-linking of¹²⁵ I-FGF9 to wild-type CHO-KI cells expressing FGFR3 was not affected by exogenous heparin. However, in the absence of heparin, no cross-linking of FGF9 to mutant HS-deficient CHO-745 cells expressing FGFR3 was detected (Fig. 7), supporting the notion that heparin-like molecules are required for efficient affinity responses of FGF9. When heparin was added, 745-FGF3 and¹²⁵ I-FGF9 affinity labels were prominent and indistinguishable from wild-type cells, which indicated that heparin could support the high-affinity binding between FGF9 and FGFR3. Binding on both cells was specific and saturable (in the presence of 1 mg/ml heparin), with kD of 0.06 and 0.1 nM for CHO-KI cells and CHO745 cells, respectively. A typical heparin dose-dependent increase was obtained when FGF9 bound to CHO 745-FGFR3 transfected cells, with maximal specific binding at approximately 500 ng/ml heparin (data not shown).

实施例5：DNA合成测定Example 5: DNA Synthesis Assay

用在24孔板中，附加10％胎牛血清的F12培养物中生长的铺满培养物可测定掺入CHO的胸苷。使细胞在无血清下饥饿24小时，然后与或不与各种浓度的FGF9或与10％血清(作为对照)再培育14小时，然后加入3H-胸苷(0.5mCi/ml)再反应2小时。在培育的最后，细胞用冷的PBS洗涤两次，用冰冷的5％三氯乙酸固定20分钟，用95％乙醇洗涤并溶解在0.1MNaOH中。用液体闪烁计数来测定DNA相关的放射性。Thymidine incorporation into CHO was determined using confluent cultures grown in F12 cultures supplemented with 10% fetal calf serum in 24-well plates. Cells were starved for 24 hours without serum, then incubated with or without various concentrations of FGF9 or with 10% serum (as a control) for an additional 14 hours, and then added 3H-thymidine (0.5mCi/ml) for an additional 2 hours . At the end of the incubation, cells were washed twice with cold PBS, fixed with ice-cold 5% trichloroacetic acid for 20 min, washed with 95% ethanol and dissolved in 0.1 M NaOH. DNA-associated radioactivity was determined by liquid scintillation counting.

为测定FGFR3的活化是否也需要肝素，我们研究了FGF9诱导表达FGFR3的HS-缺陷性CHO 745细胞内DNA合成所需的肝素量。没有外源性肝素时，则通过FGF9掺入3H-胸苷没有发现有显著的增加(图8A)，这与受体结合的缺少一致且与其它研究的FGF对肝素的严格需要相似。加入低浓度的肝素可显著地以与剂量依赖的方式促进依赖于FGF9的DNA合成，并且最大效应的一半和最大效应分别在100ng/ml和2mg/ml。单独施用肝素对DNA合成无影响，且CHO-KI中FGF9诱导的DNA合成与外源性肝素无关(未显示)。To determine whether heparin is also required for activation of FGFR3, we investigated the amount of heparin required for FGF9-induced DNA synthesis in HS-deficient CHO 745 cells expressing FGFR3. In the absence of exogenous heparin, no significant increase in 3H-thymidine incorporation by FGF9 was found (Fig. 8A), consistent with the lack of receptor binding and similar to other studies of FGF's strict requirement for heparin. The addition of low concentrations of heparin significantly enhanced FGF9-dependent DNA synthesis in a dose-dependent manner, with half and maximum effects at 100 ng/ml and 2 mg/ml, respectively. Heparin alone had no effect on DNA synthesis, and FGF9-induced DNA synthesis in CHO-KI was independent of exogenous heparin (not shown).

为研究肝素促进FGF9结合所需的结构，我们分析了一系列大小为6至18个单糖单元的肝素组分对于FGF9诱导DNA合成的效应。尽管6单体的肝素组分抑制了FGF9的效应，但是在8-10单体组分发现有DNA合成诱导，当肝素片段为14-16单体时，FGF9有最大效应(图8B)。这些结果表示FGFR3被FGF9激活需要一个特定大小的肝素。To investigate the structure required for heparin to promote FGF9 binding, we analyzed the effect of a series of heparin components ranging in size from 6 to 18 monosaccharide units on FGF9-induced DNA synthesis. Although the 6-mer heparin fraction inhibited the effect of FGF9, induction of DNA synthesis was found at the 8-10-mer fraction, with the greatest effect of FGF9 when the heparin fragment was 14-16-mer (Fig. 8B). These results indicate that activation of FGFR3 by FGF9 requires a specific size of heparin.

实施例6表达FGF9的质粒构建物Embodiment 6 expresses the plasmid construct of FGF9

为表达重组的FGF9，用细菌表达载体pET-3C的Ndel/BamH位点来亚克隆鼠FGF9的cDNA。在BL-21细胞转化与1mM IPTG诱导后，裂解细胞并在肝素-琼脂糖凝胶柱上纯化FGF9。To express recombinant FGF9, the cDNA of murine FGF9 was subcloned using the Ndel/BamH sites of the bacterial expression vector pET-3C. After transformation of BL-21 cells and induction with 1 mM IPTG, cells were lysed and FGF9 was purified on a heparin-agarose column.

将全长鼠FGF9 cDNA亚克隆人胶原IIAl基因的剪接受体位点的下游，胶原IIAl基因位于其启动子和软骨特异性增强子后。构建物被线性化，并用于注射入受精的鼠卵中以产生转基因鼠。The full-length mouse FGF9 cDNA was subcloned downstream of the splice acceptor site of the human collagen IIA1 gene behind its promoter and cartilage-specific enhancer. The constructs were linearized and used for injection into fertilized mouse eggs to generate transgenic mice.

实施例7转基因动物的FGF9过量表达Example 7 Overexpression of FGF9 in transgenic animals

转基因鼠用上述的过量表达FGF9的载体转化。这些转基因鼠与用FGFR3-Ach突变(有软骨发育不全的FGFR3突变)的转基因鼠非常相似，只是体形小，短尾和短后肢的特征。这种转基因鼠可作为各种侏儒症和由FGF9过量导致的异常症状的模型。Transgenic mice were transformed with the above-mentioned vectors overexpressing FGF9. These transgenic mice were very similar to transgenic mice with the FGFR3-Ach mutation (FGFR3 mutation with achondroplasia), but were characterized by small body size, short tail and short hindlimbs. This transgenic mouse can be used as a model for various dwarfism and abnormal symptoms caused by FGF9 excess.