CN100340573C - Heterocarpine,人GHRH结合蛋白 - Google Patents
Heterocarpine,人GHRH结合蛋白 Download PDFInfo
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- CN100340573C CN100340573C CNB028054660A CN02805466A CN100340573C CN 100340573 C CN100340573 C CN 100340573C CN B028054660 A CNB028054660 A CN B028054660A CN 02805466 A CN02805466 A CN 02805466A CN 100340573 C CN100340573 C CN 100340573C
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Abstract
本发明涉及人GHRH(人生长激素释放激素)结合蛋白。上述得自植物Pilocarpus Heterophyllus的蛋白可用于制备抗GHRH作用和治疗增生性疾病(特别是癌症)、肢端肥大症或糖尿病相关肾病和视网膜病的药剂。
Description
本发明涉及人GHRH(人生长激素释放激素)结合蛋白。
生长激素(“GH”)是含有191个氨基酸的蛋白，它刺激许多生长因子如类胰岛素生长因子I(IGF-1)的生成和大量组织如骨骼、结缔组织、肌肉和肠的生长。GH也具有生理活性，能促进核酸、蛋白质的合成与脂肪的分解，减少尿的分泌(Frohman L.A.&Kineman，R.D.，Handbook ofPhysiology，Hormonal Control of Growth，Kostyo，J.L.&Goodman，H.M.编著(Oxford Univ.Press，New York，1999)，p.189-221)。
G H的合成受到下丘脑分泌因子的正或负调节。控制GH生成的主要因子是“生长激素释放激素”，它是人体中一种含44个氨基酸的肽。
GH和GHRH和很多疾病相关。其中特别要提的是：癌症(特别是前列腺癌或肺癌)，肢端肥大症，糖尿病相关视网膜病和肾病；对于这些疾病，需要用GHRH拮抗剂治疗。由于它潜在的和这么多疾病相关，工业上一直进行GHRH拮抗剂的研究。
申请人从植物中分离出了一个新的蛋白，它能和人GHRH结合。
因此，本发明的第一个主题就是从植物Pilocarpus heterophyllus中提取的一种蛋白，其特征是它的分子量约为90.9kDa，且包含SEQ.ID.NO.1，SEQ.ID.NO.2和SEQ.ID.NO.3的肽片段。上述蛋白可以呈糖基化或非糖基化的形式。为了简化下面的公开，此后将该蛋白称为“heterocarpine“。
上述SEQ.ID.NO.1，SEQ.ID.NO.2和SEQ.ID.NO.3的序列如下：
SEQ.ID.NO.1： KLIGARYFDK
SEQ.ID.NO.2： YGEDIIVGVIDSGV
SEQ.ID.NO.3： PESESY
上面描述肽所用的术语(本申请剩下部分也会用到)为“IUPAC-IUB生物化学术语委员会”所规定，根据标准表示方式，N-端氨基酸(氨基)位于左边，C-端氨基酸(羧基)位于右边。术语“天然氨基酸”是指在自然界中发现的L-型氨基酸：Gly，Ala，Val，Leu，Ile，Ser，Thr，Lys，Arg，Asp，Asn，Glu，Gln，Cys，Met，Phe，Tyr，Pro，Trp和His。
如果一种蛋白分离自原来的环境，则其被称为“分离的”，特别是当它从与其并存于天然系统中的生物物质中分离出来时。
优选本发明涉及非糖基化形式的heterocarpine。
根据本发明优选方案，heterocarpine来自体外培养的植物PilocarpusHeterophyllus的细胞提取物。
此外，本发明的另一个主题是一种单克隆抗体，或者是该单克隆抗体的抗原结合片段，它能专一性的和heterocarpine结合。
Heterocarpine具有和人GHRH结合的特性。在体外，heterocarpine能和人RHRH结合而阻止了人GHRH和其受体结合后引起的环AMP的合成。用大鼠进行的体内实验发现，heterocarpine/人GHRH复合物在血管腔隙(blood compartment)中以摩尔与摩尔的比例形成，它以剂量依赖型方式抑制GH的合成，其中GH的合成由10μg人GHRH诱导。Heterocarpine具有和人GHRH结合的特性。
本发明化合物的这些特点使其适合用于制药。因此，本发明的再一个主题是作为药剂的糖基化或非糖基化的heterocarpine。本发明也涉及药物组合物，它含有糖基化或非糖基化的heterocarpine作为活性成分，也包含一种或更多的制药学上可接受的赋形剂。另一个主题是糖基化或非糖基化heterocarpine的应用，用来制备抗GHRH影响的药剂以治疗增生性疾病(特别是癌症)、肢端肥大症或糖尿病相关视网膜病和肾病。对于癌症，heterocarpine特别适合制备药剂，用以治疗良性肿瘤和胰腺癌，下丘脑-垂体神经节细胞癌，支气管的、肠的和肝脏的癌，交感性肾上腺素肿瘤，嗜铬细胞瘤，垂体腺瘤和甲状腺癌。
本发明也涉及作为药剂的单克隆抗体，或其能专一性的和heterocarpine结合的抗原结合片段。它也和药物组合物相关，该组合物含有作为活性成分的单克隆抗体或者能专一性的和抗原heterocarpine结合的片段，也包含一种或更多的制药学上可接受的赋形剂。该主题也涉及单克隆抗体或者能专一性的和抗原heterocarpine结合的片段的应用，用以治疗增生性疾病(特别是癌症)、肢端肥大症或糖尿病相关视网膜病和肾病。对于癌症，上述单克隆抗体或者上述专一性的和抗原heterocarpine结合的片段特别适于制药，用以治疗良性肿瘤和胰腺癌，下丘脑-垂体神经节细胞癌，支气管的、肠的和肝脏的癌，交感性肾上腺素肿瘤，嗜铬细胞瘤，垂体腺瘤和甲状腺癌。
本发明也涉及使用heterocarpine作为赋形剂用于药物组合物中以控制GHRH的释放。本发明也涉及含有GHRH，heterocarpine和一种或多种制药学上可接受的赋形剂的药物组合物。
最后，本发明其他主题是从植物Pilocarpus Heterophullus细胞中提取和分离heterocarpine的方法，优选上述细胞来自体外培养物。这些方法主要包括用水对植物Pilocarpus Heterophullus细胞的提取步骤，温度为0到50℃，优选4到25℃，上述提取步骤后为过滤步骤，以从PilocarpusHeterophullus细胞中分离出富含heterocarpine的滤出液。之后是一个或多个步骤以将heterocarpine从植物Pilocarpus Heterophullus提取液中其他组分中分离出来。
根据第一个可选方案，这些提取和分离方法主要包括下面的连续步骤：
a)用水对植物Pilocarpus Heterophullus细胞的提取步骤，温度为0到50℃，优选4到25℃，上述提取步骤后为过滤步骤，以从Pilocarpus Heterophullus细胞中分离出富含heterocarpine的滤出液；
b)抽提液中蛋白质的沉淀步骤，如加入硫酸铵后对沉淀物进行分离(通过过滤或优选通过离心)；
c)将步骤b)中回收的沉淀物溶于水中；以及
d)凝胶过滤层析步骤以将heterocarpine从含有其他组分的溶液中分离出来。
根据另一个可选方案，这些提取和分离过程主要包括如下连续步骤：
a)用水对植物Pilocarpus Heterophullus细胞的提取步骤，温度为0到50℃，优选4到25℃，上述提取步骤后为过滤步骤，以从Pilocarpus Heterophullus细胞中分离出富含heterocarpine的滤出液；
b)对a)中得到的溶液进行脱脂步骤，加入非氧化酸(如盐酸，硫酸或磷酸)使其酸化，调节pH优选2到4之间，用液-液萃取(优选为加入有机溶剂如二氯甲烷、庚烷、己烷或环己烷)；
c)去除单宁步骤，让b)中得到的脱脂溶液和聚乙烯吡咯烷酮(或尼龙66)接触，之后用大孔树脂(优选为用聚苯乙烯基树脂如树脂DiaionHP-20)过滤；
d)通过加入碱如氢氧化铵、氢氧化钠或氢氧化钾调节步骤c)所得滤液至碱性pH(优选pH在9到11之间)；
e)用阴离子交换树脂进行的一个或多个过滤步骤，这个或这些过滤步骤的洗脱液优选为缓冲液，pH在9到11之间，可选择性含有浓度梯度的盐(如氯化钠或硫酸铵)，以将heterocarpine从溶液中其他组分中分离出来；
f)脱盐步骤，将步骤e)中所得溶液过树脂，按照分子量大小进行混合物组分的分离(如树脂SephadexG25或Superdex200HR)，用水对树脂上的混合物进行洗脱。
含有本发明化合物的药物组合物可以为固态，如粉末、药丸、颗粒、药片、脂质体、明胶胶囊或栓剂。药丸、药片或明胶胶囊可以用能够保护药物不被胃酸或病人胃中的酶破坏的物质包被，以让其有足够的时间不被消化而进入病人的小肠。该化合物也可以局部用药，如用于肿瘤确切的位置。该化合物也可以用缓释方法给药(如用缓释组合物或灌注泵)。合适的固态载体可为如磷酸钙、硬脂酸镁、碳酸镁、滑石粉、蔗糖、乳糖、糊精、淀粉、白明胶、纤维素、甲基纤维素、羧甲基纤维素钠、聚乙烯吡咯烷酮和蜡。
含有本发明化合物的药物组合物也可以为液态，如溶液、乳液、悬浮液或缓释制剂。适宜的液态载体可以是，如水，有机溶剂如甘油或二元醇如聚乙二醇，以及它们在水中的不同比例的混合物。
用以治疗上述疾病的本发明的化合物剂量，按照给药方法、所治疗对象的年龄和体重及状态而有所变化，且最终由有关医生或兽医决定。这种由相关医生或兽医决定的用量在这儿称作“有效治疗用量”。
根据本发明，可按照后面所述方法制备heterocarpine。
制备heterocarpine
根据本发明的一个优选方案，体外培养的愈伤组织或细胞悬浮物来自植物不同器官。这些培养于半固体或液体培养基上的组织能够生物合成具生物学性质的化合物。本申请中的“愈伤组织”指半固体营养培养基上的未分化的肉眼可见的细胞团。本申请中术语“未分化细胞”指能够在一定条件下以愈伤组织或细胞悬浮物的形式增殖的但没有形态发生现象的细胞。最后，所谓“细胞悬浮物”，指未分化的细胞，它们能在液体营养培养基中形成显微镜下可见的团块。
营养培养基、激素、培养条件的选择以及这些体外培养物的提取和对提取物进行的分析形成本发明的一个整体部分。
来自pilocarpus heterophullus种子的细胞可如根据后面的方法悬浮培养。
培养前，对其器官按照常用方法消毒。胚芽器官也可以作为愈伤组织生成起始物质进行体外培养而不用事先消毒。优选的基本营养培养基是常用的体外培养基：Gamborg培养基(见Gamborg等，Nutrient requirementsof suspension cultures of Soybean root cells，Exp.Cell Res.(1968)，50(1)，151-158)。碳源为蔗糖但也可使用浓度为1到120g/l，优选约30g/l的葡萄糖。大组分的含量也可减少一半。加入生长素或一半生长素一半细胞分裂素，优选为两种激素联合使用，通常2，4-二氯苯氧乙酸和激动素，但α-萘乙酸(NAA)，β-吲哚乙酸(IAA)，β-吲哚丁酸(IBA)或毒莠定也可以和激动素或苄氨基嘌呤(BAP)联合使用。生长素浓度可以在0.1到10mg/l之间(如可选择1mg/l)，细胞分裂素浓度可以在0.01到2mg/l之间(如可选择0.06mg/l)。在不同的基本培养基中要加入相应维生素。在光或黑暗下进行培养。温度可在10到33℃但优选约为23℃。培养基pH在4到6.5之间，优选为在灭菌之前调节至pH 5.8。而且，在培养基中可或不加入琼脂。
几天后培养基中出现初生愈伤组织，可以将它从原来的植入物中分离出来，再次培养约1个月然后在琼脂半固体培养基(在试管或培养皿中)培养4到8周，优选6周，然后愈伤组织可以通过在新的培养基上连续传代培养而保持几年。愈伤组织也可以在摇动的液体培养基(锥形瓶或生物反应器)中传代培养2到6个周，优选为3周。
所得株系可由它们的遗传来源、培养条件、外观和缺乏形态发生来分辨。
冻干的Pilocarpus Heterophyllus细胞在0到50℃，优选4到25℃下用水提取。然后将所得提取物冻干，重新溶解到合适的浓度(如大约含30％的干物质)。加入浓硫酸铵溶液(如代表70到90％饱和度的浓度)，沉淀蛋白用最少量的水溶解，不溶物质通过离心回收。将蛋白用柱层析(洗脱液优选为水)进行分离，然后可将heterocarpine(可通过其分子量约为90.0kDa来鉴定)回收。
制备和heterocarpine特定结合的抗体
本发明提供和heterocarpine特定结合的制剂如抗体。这种“特定结合”某个蛋白的制剂是指它能和上述蛋白的反应在可检测的水平上(如通过酶联免疫吸附测定法)且不和其他蛋白有可检测出的反应。“结合”是指两个单独的分子间的非共价键结合而形成复合物。可以，如通过测定形成复合物的结合常数来估计结合的能力。结合常数为复合物浓度与非复合物成分浓度乘积的比值。当结合常数达到103l/mol时，两个产物称为是“结合”的。结合常数的测定可以使用该领域熟练人员所熟知的方法。
满足上述标准的任何制剂都可认为是结合制剂。
本发明中，结合制剂优选为抗体或抗体的一个片段。抗体的制备可以用该领域熟练人员所知的任何技术(参照Harlow and Lane，Antibodies：Alaboratory Manual，Cold Spring Harbor Laboratory，1988)。通常，可以通过细胞培养技术包括制备单克隆抗体或通过将抗体基因转染到来自细菌或哺乳动物中的宿主细胞中以生产重组抗体。其他技术中，优选使用后面所描述的方法。将含有heterocarpine的免疫原注射到一组哺乳动物(如小鼠、大鼠、家兔、绵羊或山羊)中。在这个阶段，heterocarpine可作为没有修饰的免疫原。或者，如果heterocarpine和转运蛋白如牛血清白蛋白或帽贝血蓝蛋白联合使用可以引起更强的免疫反应。优选根据预定的时间表，将免疫原注射到宿主动物，对宿主动物定期取血。Heterocarpine特定结合多克隆抗体可以从这种抗血清中纯化出来，如通过将heterocarpine与合适的固体载体连接后进行亲和层析。
用于释放GHRH的药物组合物：
特别的，可以根据期刊Pharmacological Reviews(2000)，52，207-236中De Wolf和Brett所描述的方法以及其中所引用的文献中所描述的方法之一，用heterocarpine和GHRH制备药物组合物。
除非另外特别说明，这里所用的所有科学术语的意思都和本发明所属领域普通专业人员所通常理解的意思相同。类似的，这里所提到的出版物、专利申请、所有专利和所有其他文献都并入本文作为参考。
下面的实施例用于阐明上面的方法但决不能认为是对本发明范围的限制。
获得heterocarpine
实施例1：
细胞的体外培养：
使Pilocarpus Heterophyllus种子发芽，去除发芽种子的茎干。上述茎干培养于Gamborg培养基(Gamborg等，Nutrient requirements ofsuspension cultures of Soybean root cells，Exp.Cell Res.(1968)，50(1)，151-158)中，其中已加入30g/l的蔗糖，1mg/l的2，4-二氯苯氧乙酸和0.06mg/l的激动素。在温度23℃，黑暗中用试管进行培养。在通常条件下，每6周进行传代培养。株系外观呈粒状，有浅褐色的色素沉着。
基于生物量的鲜和干物质的增加，测定株系的生长动力学八周以上。合并两管中的愈伤组织作为每周两次的收获，第一次收获发生时间为0。将愈伤组织和琼脂糖收获后冻干。可以看到直到第6周都呈指数生长，然后出现静止生长期。
细胞培养物的提取：
取25g冻干的Pilocarpus Heterophyllus细胞浸入375ml 4℃水中提取两次，于4℃过夜，然后置于250ml 4℃水中4小时，最后在4℃用125ml水冲洗。所得水溶液都在真空下通过覆盖有硅藻土的玻璃滤器过滤以将细胞碎片从水溶液中除去。将水溶液合并后冻干得到9.4g干物质。将冻干后的抽提物溶于31ml 20℃水中，得到含30％干提取物的溶液。缓慢加入17.4g硫酸铵并用磁力搅拌器不断搅动以沉淀蛋白组分。在3000rpm下离心20分钟以将蛋白沉淀物从硫酸铵溶液中分离出来。倒出硫酸铵溶液，用22ml水溶解沉淀的蛋白，再次离心、过滤以除去不溶性物质。
将滤液进行凝胶过滤层析。将滤液注入到根据厂家推荐方法制作的装满SuperdexTM200(Amersham Pharmacia Biotech，reference no.17-1043-01；颗粒直径平均为13μm)的柱子(Buchi N019678，长度＝230mm；内径＝26mm)中，用超纯水(Water’s Milli-Q)作为洗脱液，流速5ml/分钟。收集到40ml组分并在第三和第四组分中发现活性蛋白。将这些组分冻干得到约14.2mg的活性产物。
对产物用含十二烷基硫酸钠的凝胶电泳(SDS PAGE)检验发现为一条带，证明产物已纯。以后把对应于这条带的产物叫做heterocarpine。
实施例2：
用与实施例1中所描述相同的方法体外培养的细胞按照后面所描述的方法进行提取。在20℃将100g冻干的Pilocarpus Heterophyllus细胞用2升去离子水提取，将混合物在搅拌下过夜。细胞和提取物通过在覆盖有硅藻土层(先前用酸洗过，1到2lm厚)的玻璃料(孔隙率3，直径20lm)上抽气进行过滤。在去除前将回收的细胞用400ml去离子水冲洗。加入约10ml 18％的盐酸，将滤液酸化至pH 3.0。酸性溶液通过加入400ml二氯甲烷液-液萃取以脱脂。倒出并除去二氯甲烷相。将脱脂的溶液进行旋转蒸发以除去剩余的二氯甲烷。加入约30g聚乙烯吡咯烷酮到脱脂溶液中(pH约3.0)并搅拌约30分钟以除去单宁酸。将混合物用覆盖有由25g硅藻土(先用酸洗过)和25g聚乙烯吡咯烷酮组成的混合层的玻璃料(孔隙率3，直径10lm)抽气过滤。滤液穿过根据厂家说明书预先活化的400mlDiaionHP-20(Mitsubishi Chemical Company)床。所得滤液加入约60ml20％氢氧化铵溶液调节pH为碱性(pH10)。静置30分钟后出现少量沉淀。往碱性溶液中加入1g硅藻土(事先用酸洗过)，然后通过膜过滤器(0.22μm)抽气过滤。将约2升滤液穿过HiPrepQXL 16/10柱，该柱装在Akta纯化器中并事先用0.1M哌嗪/盐酸缓冲液平衡到pH 10.2，流速为0.5ml/分钟(HiPrepQ XL 16/10柱和Akta纯化器都是AmershamBiosciences公司产品)。柱子用6个柱体积pH10.2的起始缓冲液、5个柱体积的含0.2M氯化钠的相同缓冲液、10个柱体积的含1M氯化钠的相同缓冲液进行连续洗脱。回收的大多数heterocarpine都在含1M氯化钠溶液的前3个柱体积中。将活性组分用SephadexG25脱盐(柱床体积：260ml)，用去离子水作为洗脱液。将相应与滞留体积的第一个柱体积中的活性组分冻干，得到170mg heterocarpine。所得heterocarpine在SDSPAGE凝胶上几乎是一条带。
Heterocarpine的鉴定
分析与微序列分析：
将样品上到10％聚丙烯酰胺凝胶中。迁移后，将凝胶固定并用考马斯亮蓝染色。
图3表示的凝胶道相应的道1，2，3，4和5分别为分子量标记，0.5、1和2μg实施例1中所得最终heterocarpine组分和分子质量标记(Amersham)。通过标准分子质量标记图，利用本领域熟练人员所熟知的标准计算工具(如，Viber Lourmat’s Bio-Profil Bio1D软件)测定分子量，测得heterocarpine分子质量为90.9K道尔顿(±1.6K道尔顿)。
为了进行蛋白质微序列分析，将含有蛋白质的聚丙烯酰胺带切下并用300μl含50mM的Tris(pH 8.6)，0.03％十二烷基硫酸钠的消化缓冲液，在0.4μg内溶素-C(Sigma)存在的条件下于35℃消化18小时。所得肽用H PLC分离，所用柱子为直径1mm D EAE-C18内置柱。分离梯度基于乙腈(从2到70％)和0.1％三氟乙酸(TFA)。在Procise测序仪(Applied Biosystem)上测序。通过这种方法，测定了三个峰的序列，使得可能用独特的方法描述heterocarpine的特征。相应序列在本发明中用SEQ.ID.NO.1，SEQ.ID.NO.2和SEQ.ID.NO.3标识。
糖蛋白的分析通过检测用SDS-PAGE凝胶所分开的糖蛋白的糖结构。该检测系统根据“Periodic Acid-Schiff”方法修改，出现紫红色的带表明存在糖蛋白(Sigma)。对于实施例1中所得heterocarpine，在图4中为所复制的结果。
Heterocarpine的药理学特性
人GHRH受体(hGHRH-R)的稳定转染：
能稳定的表达人GHRH受体的人胚胎肾脏细胞，HEK-293，(细胞系由纽约Mount Sinai Medical School的Stuart Sealfon博士开发)得自KellyMayo博士(Northwestern University，Chicago，IL)。
细胞培养和膜的制备：
将用上述人GHRH受体稳定转染的HEK-293细胞培养于DMEM(Dulbecco’s modified Eagle’s medium，高葡萄糖成分；由Lifetechnologies提供)中，其中培养基中补充有0.4mg/ml G418(Lifetechnologies)，10％胎牛血清和4mM L-谷酰胺(Life technologies)。将细胞在缓冲液A中匀浆，其中缓冲液A含有50mM HEPES(pH 7.4)，5mM氯化镁(MgCl2)，2mM 乙二醇-二(2-氨基-乙基)-N，N，N’，N’-四乙酸(EGTA)和50μg/ml杆菌肽，然后在缓冲液A中对细胞进行超声破碎。匀浆后的细胞在4℃以39,000g离心10分钟，悬浮于缓冲液A中，然后在4℃以40,000g再次离心10分钟。总膜蛋白用Bradtord法定量。沉淀后的细胞膜保存在-80℃备用。
hGHRH-R的竞争性结合试验：
将用人GHRH受体稳定转染的HEK-293细胞膜在反应缓冲液中稀释到100μg/ml，其中反应缓冲液含有50mM HEPES(pH7.4)，5mM MgCl2，2mM EGTA，50μg/ml杆菌肽和0.5％的小牛血清(BSA)。将膜和0.05nmol[125I]GHRH(1-44酰胺)(Amersham)在23℃孵育2小时，其间增加heterocarpine浓度，终体积为200μl。通过0.1％预装聚氮丙啶的96孔G F/C过滤器快速过滤来终止反应。将过滤器在4℃利用Packard 96孔过滤装置用含50mM Tris(pH 7.4)的洗涤缓冲液洗3次。将过滤器干燥后浸入20μl闪烁鸡尾酒(Miroscint O，Packard)然后进行Topcount计数(Packard)。非特异性活力的测定在存在100nM的hGHRH的条件下进行。对hGHRH(0.001nM-100nM)作剂量反应曲线，所得结果见图1。
环AMP的竞争性生成：
将用人GHRH受体稳定转染的HEK-293细胞分散于48孔培养板中并培养3天。除去培养基后用培养基B代替，其中含250μl DMEM(Dulbecco’s modified Eagle’s medium，高葡萄糖成分；由Life technologies提供)，0.5％BSA，0.5mM 3-异丁基-1-甲基黄嘌呤(IBMX)，在37℃预孵育5分钟。在预孵育阶段的最后，用20分钟测定heterocarpine。所测浓度见图2。加入100μl 0.1M的HCl终止反应，用FlashPlate试剂盒(New England Nuclear)分析等分部分环AMP的含量。
分析大鼠中GH：
用Spi-Bio(Spi-Bio，法国)开发的酶-免疫试验测定大鼠(SpragueDawley雄性)血样中的GH水平。对大鼠静脉注射heterocarpine并增加剂量(仅赋形剂，1，3和10mmol)，10分钟后注射10μg(3nmol)hGHRH。注射hGHRH 10分钟后，用上述方法测量血样中激素增长水平。所得结果见图5。
附图简述
图1所示为随着heterocarpine浓度的增加，GHRH结合人GHRH受体受到抑制。
图2所示为随着heterocarpine浓度的增加，在10nM人GHRH存在条件下，人GHRH受体稳定转染的细胞中环AMP的生成受到抑制。
图3为SDS-PAGE蛋白质凝胶板的复制品，显示heterocarpine的分子量为90.9kDa。
图4为SDS-PAGE蛋白质凝胶板的复制品，显示heterocarpine为糖蛋白(面板B)。
图5所示柱状图表示随着heterocarpine浓度的增加，在10μg人GHRH存在时，大鼠中GH的合成受到抑制。
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Claims (12)
1.从植物Pilocarpus heterophyllus中提取的人GHRH结合蛋白质heterocarpine，其特征是它的分子量为90.9kDa±1.6kDa，并包含肽序列SEQ.ID.NO.1，SEQ.ID.NO.2和SEQ.ID.NO.3；上述蛋白可以呈糖基化或非糖基化的形式。
2.根据权利要求1的蛋白质，其特征是它从体外培养的植物Pilocarpus heterophyllus细胞提取物中得到。
3.权利要求1或2的蛋白质在制备用于治疗与GHRH相关的疾病的药物中的用途。
4.单克隆抗体或者其抗原结合片断，它特异性地和权利要求1中的人GHRH结合蛋白质结合。
5.权利要求4的单克隆抗体或其抗原结合片断在制备用于治疗与GHRH相关的疾病的药物中的用途。
6.用于治疗与GHRH相关的疾病的药物组合物，包含作为活性成分的根据权利要求1或2的蛋白质和一种或多种药物学上可接受的赋形剂。
7.根据权利要求1或2的蛋白质在制备抗GHRH作用的药物中的用途。
8.根据权利要求1或2的蛋白质在制备治疗与GHRH相关的增殖性疾病的药物中的用途。
9.根据权利要求8的用途，其中的增殖性疾病为癌症。
10.根据权利要求1或2的蛋白质在制备治疗与GHRH相关的肢端肥大症的药物中的用途。
11.根据权利要求1或2的蛋白质在制备治疗与GHRH相关的糖尿病相关视网膜病和肾病的药物中的用途。
12.从植物Pilocarpus heterophyllus细胞中提取和分离heterocarpine的方法，它包括在0到50℃用水对植物Pilocarpusheterophyllus细胞进行提取的步骤，上述提取步骤之后为过滤步骤，将富含heterocarpine的滤液与Pilocarpus heterophyllus细胞分开，以及一个或多个分离步骤，将heterocarpine从植物Pilocarpusheterophyllus提取物其他组分中分离出来。
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| FR01/02631 | 2001-02-27 | ||
| FR0102631A FR2821359B1 (fr) | 2001-02-27 | 2001-02-27 | L'heterocarpine, une proteine fixant le ghrh humain |
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| CN1531550A CN1531550A (zh) | 2004-09-22 |
| CN100340573C true CN100340573C (zh) | 2007-10-03 |
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|---|---|---|---|
| CNB028054660A Expired - Fee Related CN100340573C (zh) | 2001-02-27 | 2002-02-26 | Heterocarpine,人GHRH结合蛋白 |
Country Status (20)
| Country | Link |
|---|---|
| US (1) | US7385024B2 (zh) |
| EP (1) | EP1409531B1 (zh) |
| JP (1) | JP4117194B2 (zh) |
| KR (1) | KR20030094262A (zh) |
| CN (1) | CN100340573C (zh) |
| AT (1) | ATE309269T1 (zh) |
| AU (1) | AU2002241065B2 (zh) |
| BR (1) | BR0207578A (zh) |
| CA (1) | CA2437516A1 (zh) |
| CZ (1) | CZ20032612A3 (zh) |
| DE (1) | DE60207257T2 (zh) |
| ES (1) | ES2252431T3 (zh) |
| FR (1) | FR2821359B1 (zh) |
| HU (1) | HU225431B1 (zh) |
| IL (2) | IL157150A0 (zh) |
| MX (1) | MXPA03007626A (zh) |
| NZ (1) | NZ527749A (zh) |
| PL (1) | PL207581B1 (zh) |
| RU (1) | RU2305683C2 (zh) |
| WO (1) | WO2002068461A2 (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2843697B1 (fr) * | 2002-08-26 | 2005-12-09 | Sod Conseils Rech Applic | L'heterocarpine, une proteine d'origine vegetale aux proprietes anticancereuses |
| US7494969B2 (en) | 2002-02-26 | 2009-02-24 | Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) | Heterocarpine, a plant-derived protein with anti-cancer properties |
| CN1688696A (zh) * | 2002-09-18 | 2005-10-26 | 蒙特利尔大学医疗中心 | Ghrh类似物 |
| FR2848221B1 (fr) * | 2002-12-10 | 2007-09-28 | Sod Conseils Rech Applic | Procede de preparation d'heterocarpine recombinante |
| JP2005289895A (ja) * | 2004-03-31 | 2005-10-20 | Kagoshima Tlo Co Ltd | 植物タンパク質分離精製法及びそれに用いるタンニン中和剤並びにその調製法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995016707A1 (en) * | 1993-12-17 | 1995-06-22 | The Administrators Of The Tulane Educational Fund | ANALOGUES OF hGH-RH (1-29)NH2 HAVING ANTAGONISTIC ACTIVITY |
| EP0674007A1 (fr) * | 1994-03-21 | 1995-09-27 | Societe Des Produits Nestle S.A. | Procédé de production de pilocarpine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050160088A1 (en) | 2001-05-17 | 2005-07-21 | Todd Scallan | System and method for metadata-based distribution of content |
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2001
- 2001-02-27 FR FR0102631A patent/FR2821359B1/fr not_active Expired - Fee Related
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2002
- 2002-02-26 RU RU2003128963/13A patent/RU2305683C2/ru not_active IP Right Cessation
- 2002-02-26 CN CNB028054660A patent/CN100340573C/zh not_active Expired - Fee Related
- 2002-02-26 ES ES02706900T patent/ES2252431T3/es not_active Expired - Lifetime
- 2002-02-26 AU AU2002241065A patent/AU2002241065B2/en not_active Ceased
- 2002-02-26 IL IL15715002A patent/IL157150A0/xx active IP Right Grant
- 2002-02-26 AT AT02706900T patent/ATE309269T1/de not_active IP Right Cessation
- 2002-02-26 NZ NZ527749A patent/NZ527749A/en unknown
- 2002-02-26 EP EP02706900A patent/EP1409531B1/fr not_active Expired - Lifetime
- 2002-02-26 CA CA002437516A patent/CA2437516A1/fr not_active Abandoned
- 2002-02-26 CZ CZ20032612A patent/CZ20032612A3/cs unknown
- 2002-02-26 WO PCT/FR2002/000691 patent/WO2002068461A2/fr active IP Right Grant
- 2002-02-26 MX MXPA03007626A patent/MXPA03007626A/es active IP Right Grant
- 2002-02-26 DE DE60207257T patent/DE60207257T2/de not_active Expired - Lifetime
- 2002-02-26 JP JP2002567971A patent/JP4117194B2/ja not_active Expired - Fee Related
- 2002-02-26 PL PL369469A patent/PL207581B1/pl unknown
- 2002-02-26 US US10/470,112 patent/US7385024B2/en not_active Expired - Fee Related
- 2002-02-26 HU HU0303244A patent/HU225431B1/hu not_active IP Right Cessation
- 2002-02-26 BR BR0207578-4A patent/BR0207578A/pt not_active Application Discontinuation
- 2002-02-26 KR KR10-2003-7011194A patent/KR20030094262A/ko not_active Application Discontinuation
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2003
- 2003-07-29 IL IL157150A patent/IL157150A/en not_active IP Right Cessation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995016707A1 (en) * | 1993-12-17 | 1995-06-22 | The Administrators Of The Tulane Educational Fund | ANALOGUES OF hGH-RH (1-29)NH2 HAVING ANTAGONISTIC ACTIVITY |
| EP0674007A1 (fr) * | 1994-03-21 | 1995-09-27 | Societe Des Produits Nestle S.A. | Procédé de production de pilocarpine |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4117194B2 (ja) | 2008-07-16 |
| HU225431B1 (en) | 2006-11-28 |
| IL157150A (en) | 2008-11-26 |
| US7385024B2 (en) | 2008-06-10 |
| PL369469A1 (en) | 2005-04-18 |
| FR2821359B1 (fr) | 2003-05-09 |
| ATE309269T1 (de) | 2005-11-15 |
| PL207581B1 (pl) | 2011-01-31 |
| BR0207578A (pt) | 2004-03-02 |
| JP2004538254A (ja) | 2004-12-24 |
| IL157150A0 (en) | 2004-02-08 |
| HUP0303244A2 (hu) | 2003-12-29 |
| RU2305683C2 (ru) | 2007-09-10 |
| EP1409531B1 (fr) | 2005-11-09 |
| CZ20032612A3 (cs) | 2004-08-18 |
| EP1409531A2 (fr) | 2004-04-21 |
| MXPA03007626A (es) | 2003-12-04 |
| WO2002068461A2 (fr) | 2002-09-06 |
| FR2821359A1 (fr) | 2002-08-30 |
| HUP0303244A3 (en) | 2005-11-28 |
| US20040063621A1 (en) | 2004-04-01 |
| DE60207257T2 (de) | 2006-07-27 |
| CN1531550A (zh) | 2004-09-22 |
| WO2002068461A3 (fr) | 2004-02-12 |
| NZ527749A (en) | 2006-09-29 |
| KR20030094262A (ko) | 2003-12-11 |
| CA2437516A1 (fr) | 2002-09-06 |
| ES2252431T3 (es) | 2006-05-16 |
| RU2003128963A (ru) | 2005-02-20 |
| AU2002241065B2 (en) | 2006-09-28 |
| DE60207257D1 (de) | 2005-12-15 |
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