CN116606365A - 一种gdnf新型变体及其应用 - Google Patents
一种gdnf新型变体及其应用 Download PDFInfo
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- CN116606365A CN116606365A CN202310160825.9A CN202310160825A CN116606365A CN 116606365 A CN116606365 A CN 116606365A CN 202310160825 A CN202310160825 A CN 202310160825A CN 116606365 A CN116606365 A CN 116606365A
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Abstract
本发明涉及一种人GDNF变体,其中所述人GDNF变体的氨基酸序列相较于成熟人野生型GDNF的氨基酸序列包含以下氨基酸突变:L48A、I82V、K96P、A100P、L111A和H126R。所述人GDNF变体与野生型GDNF相比具有相似的神经保护和修复功能,但在长期施用后引发较低的代偿作用,因而更适合进行长期给药。
Description
技术领域
本发明涉及医药领域,特别是治疗性蛋白的领域。具体地,本发明涉及人胶质细胞源性神经营养因子(GDNF)的新型变体。GDNF的新型变体可用于帕金森病的治疗。
背景技术
GDNF是公知的神经营养因子,据报道,其在体外和体内向多巴胺能神经元提供营养支持。此外,已报道,GDNF在帕金森病的啮齿动物和灵长类动物模型中提供功能性改善并具有神经保护和修复作用。已将来自大肠杆菌的野生型GDNF蛋白经中枢给药至患有帕金森病的患者,并获得了一定的治疗效果。
然而,据报道,GDNF的长期持续过度表达会引起机体的代偿作用,从而导致一系列问题,包括异常出芽、酪氨酸羟化酶下调和体重减轻等。因此,临床上有必要通过调整GDNF的剂量和活性以实现更好的临床结果。
发明内容
为了解决上述问题,本发明提供了一种新的人GDNF变体,其与野生型GDNF相比具有相似的神经保护和修复功能,但在长期施用后不太容易引发机体的代偿作用,因而更适合进行长期给药。
因此,在一个方面,本发明提供了一种人GDNF变体,其中所述人GDNF变体的氨基酸序列相较于成熟人野生型GDNF的氨基酸序列包含以下氨基酸突变:L48A、I82V、K96P、A100P、L111A和H126R。
更具体地,所述人GDNF变体包含SEQ ID NO:2的氨基酸序列。编码所述人GDNF变体的核苷酸序列如SEQ ID NO:1所示。
更具体地,所述成熟人野生型GDNF包含SEQ ID NO:4的氨基酸序列。编码所述成熟人野生型GDNF的核苷酸序列如SEQ ID NO:3所示。
如本文所用,氨基酸突变L48A是指以成熟人野生型GDNF的氨基酸序列SEQ ID NO:4为参考,位置48处的亮氨酸(L)被替换为丙氨酸(A)。此定义同样适用于氨基酸突变I82V、K96P、A100P、L111A和H126R。
更具体地,所述人GDNF变体能够与GFRα1受体结合。
更具体地,所述人GDNF变体能够诱导RET磷酸化。
更具体地,所述人GDNF变体与成熟人野生型GDNF相比更慢地且以更低的水平刺激RET信号传导。
更具体地,所述人GDNF变体与成熟人野生型GDNF相比更慢地且以更低的水平刺激RET下游的MAPK途径信号传导。
更具体地,所述人GDNF变体与野生型GDNF相比具有相似的神经保护和修复功能。
更具体地,所述人GDNF变体与野生型GDNF相比,在长期施用后引发较低的代偿作用。
如本文所用,长期施用是指GDNF的长期连续施用。长期施用可以指至少1周、2周、3周、4周、1个月、2个月、3个月、4个月、5个月、6个月、一年或更久的施用。优选地,长期施用可以指至少3个月的施用。
在另一个方面,本发明提供了一种多核苷酸,其包含编码本发明的人GDNF变体的核苷酸序列。编码本发明的人GDNF变体的核苷酸序列如SEQ ID NO:1所示。
在另一个方面,本发明提供了一种病毒载体,其包含与如上文所述的多核苷酸。
更具体地,所述病毒载体选自重组痘苗病毒、重组腺病毒、重组逆转录病毒、重组腺相关病毒、重组杆状病毒、重组***瘤病毒、重组慢病毒或重组禽痘病毒。
在另一个方面,本发明提供了一种细胞,其包含如上文所述的多核苷酸或如上文所述的病毒载体。
在另一个方面,本发明提供了一种药物组合物,其包含如上文所述的人GDNF变体、多核苷酸、细胞,以及一种或多种药学可接受的稀释剂、载体或赋形剂。
更具体地,所述药物组合物用于长期给药以治疗帕金森病。
更具体地,所述药物组合物在长期给药后基本上不引起机体的代偿作用。
更具体地,长期给药可以指至少1周、2周、3周、4周、1个月、2个月、3个月、4个月、5个月、6个月、一年或更久的给药。优选地,长期给药可以指至少3个月的给药。
更具体地,所述药物组合物在长期给药后与成熟人野生型GDNF的长期给药相比引起较低的代偿作用。
更具体地,代偿作用可以包括例如多巴胺(DA)和/或酪氨酸羟化酶(TH)的表达和/或活性的下调。
在另一个方面,本发明提供了如上文所述的人GDNF变体、多核苷酸、病毒、细胞或药物组合物在制备用于长期给药以治疗帕金森病的药物中的用途。
本发明的有益效果
据报道,长期未受控制和持续的GDNF过表达会导致代偿性变化(Georgievska等人,2002b;Barroso-Chinea等人,2016),可能超过营养作用。在临床前模型中,GDNF长期连续给药后出现代偿作用,包括降低DA生物合成途径酶的表达、降低TH的转录和活性(Georgievska等人,2002a;Chtarto等人,2007)以及DAT活性(Barroso-Chinea等人,2016)。这些神经化学效应可能干扰神经营养作用,并可能降低临床获益。
本发明人通过大量的实验,惊讶地发现一种新的人GDNF变体,其通过几个特定氨基酸的突变,在不改变GDNF与其受体结合的情况下温和调控下游信号传导途径,使得既不显著改变GDNF的神经保护和修复功能,又能在更长的时期内发挥作用而不引发不利的代偿效应。不受理论约束,据信其原理在于本发明选择的氨基酸突变位点并没有在GDNF与其GFRα1受体的结合界面上,因而不影响其结合以及下游的RET磷酸化,但是本发明的突变通过改变GDNF同二聚体的结构而使得部分掩盖了GDNF-GFRα1-RET复合物中磷酸化的RET的下游信号传导结构域,导致其下游信号传导速度变得适当温和,以在不显著改变GDNF的神经保护和修复功能的情况下能够在更长的时期内发挥作用而不引发不利的代偿效应。
附图说明
图1显示了如本说明书实施例中所制备的人GDNF变体和成熟野生型人GDNF蛋白的还原性和非还原性SDS-PAGE结果。
图2显示了本发明的人GDNF变体和成熟野生型人GDNF的体外RET磷酸化ELISA检测结果。
图3显示了本发明的人GDNF变体和成熟野生型人GDNF对RET信号传导的影响。
图4显示了本发明的人GDNF变体和成熟野生型人GDNF在长期体内给药后的代偿作用。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1:人GDNF变体和成熟野生型人GDNF的制备
合成成熟野生型人GDNF(SEQ ID NO:3)和人GDNF变体(SEQ IDNO:1)的基因序列,构建到载体pET-30a(+)上,得到重组质粒。
将得到的重组质粒导入工程菌株BL21(DE3)中,得到重组菌。
将得到的重组菌接种至TB液体培养基,37℃、230rpm振荡培养至OD600=0.8-1.2,加入IPTG并使其浓度为0.5mM,然后37℃、230rpm振荡培养4h,然后8000rpm离心5min并收集菌体,将其悬浮在2-3体积的0.2mg/ml溶菌酶、5mM MgCl2和50mM Tris-Cl(pH 8)的溶液中,并使其在搅拌下在冰上孵育30分钟。将所得浆料在冰上超声处理10分钟(5秒脉冲,2秒间隔,30-40%的幅度)。此后,回收包涵体形式的人GDNF变体/成熟野生型人GDNF,通过以20000g离心20分钟将其从细胞裂解物分离并溶解在4M胍、90mM半胱氨酸、20mM Tris-Cl(pH8.5)中。通过使用0.2M胍、2M尿素、20mM Tris-Cl(pH 8.75)的10倍稀释,使蛋白再折叠成活性形式。将再折叠的混合物在4℃保持2天。
纯化:通过3步柱色谱将再折叠的人GDNF变体/成熟野生型人GDNF纯化至同质:
1.SP柱上的阳离子交换色谱(CEX)
2.苯基柱上的疏水相互作用色谱(HIC)
3.Superdex-75柱上的尺寸排阻色谱(SEC)。
首先,将复性的蛋白上样至在20mM乙酸钠(pH 5)中平衡的SP Sepharose快速流动柱。使用20mM乙酸钠(pH 5)中从0.3至1M NaCl的线性递增盐梯度洗脱人GDNF变体/成熟野生型人GDNF。向CEX主流合并物(mainstream pool)补充NaCl至2.5M的终浓度,随后上样至20mM柠檬酸钠(pH 5)中的苯基Sepharose HP疏水相互作用色谱柱。使用20mM柠檬酸钠(pH5)中从2.5至0M NaCl的线性递减盐梯度洗脱HIC柱。人GDNF变体/成熟野生型人GDNF与HIC柱紧密结合。随后,浓缩HIC主流合并物,并最后上样至Superdex-75柱,并使用PBS(pH 7.4)洗脱蛋白。浓缩最后的合并物,通过0.22微米膜过滤并在-80℃贮存。用还原和非还原SDS-PAGE鉴定人GDNF变体/成熟野生型人GDNF,并用考马斯蓝染色进行染色,显示人GDNF变体和成熟野生型人GDNF均为15.2kda亚基的同二聚体(图1)。
实施例2:人GDNF变体和成熟野生型人GDNF均能够与GFRα1受体结合
GDNF及其典型受体酪氨酸激酶RET在与GFRα1结合后的神经营养信号对在细胞培养实验和动物模型中多巴胺能神经元的存活和生理机制具有重要意义。帕金森病的特点是中脑黑质中多巴胺能细胞的死亡,这促使将GDNF/GFRα1/RET信号传导作为治疗此病的一种方法。
在ELISA测试中检验人GDNF变体和成熟野生型人GDNF与GFRα1受体的结合。使用“无GDNF”条件和/或“无关受体”条件作为阴性对照。
使用70μl的碳酸盐缓冲剂(pH 9.6)中的1μg/ml的人GFRα1(重组人GFRα1-Fc嵌合体,R&D Systems)包被96孔板(Greiner 655081免疫结合ELISA板)的每个孔。如果包被无关受体,则该无关受体是无关的Fc嵌合体,并且以与GFRα1相同的浓度包被。将板密封并在4℃孵育过夜。使用自动板清洗器,对孔进行抽吸,并用清洗缓冲剂(20mM Tris(羟甲基)氨基甲烷,pH 7.4,0.15M NaCl,0.1%Tween-20)清洗2次。在室温下,使用每孔200μl封闭缓冲剂(上述清洗缓冲剂中3%的Carnation速溶奶粉)封闭板至少1小时。用清洗缓冲剂清洗平板2次。
将GDNF蛋白在封闭缓冲剂中连续稀释到适当的浓度范围,通常从5μg/ml开始并以1:10连续稀释。使用由单独的封闭缓冲剂组成的无GDNF对照。将50μl的每种GDNF溶液添加到GFRα1包被的孔,一式三份。将板在室温下孵育1.5小时。随后,用清洗缓冲剂清洗孔3次。
向每个孔添加在封闭缓冲剂中稀释至1μg/ml浓度的生物素化抗人GDNF抗体(R&DSystems,生物素化山羊抗人GDNF多克隆抗体,目录号#BAF212)的50μl等分试样,并在室温孵育45分钟。随后,用清洗缓冲剂清洗孔3次。
向每个孔添加以1:1000稀释在封闭缓冲剂中的辣根过氧化物酶-缀合的链霉亲和素(Jackson ImmunoResearch,目录号#016-030-084)的50μl等分试样,并在室温孵育20-30分钟。或者,可以使用1:2000的稀释度,以及30-90分钟的孵育时间。随后,用清洗缓冲剂清洗孔3次。向每个孔添加50μl的显色底物(即,OPD底物),并允许其在室温显色2-3分钟。通过向每个孔添加100μl的1N HCl终止反应。在Molecular Devices SpectraMax250板读数器上读取所述孔在490nm的吸光度。测定每种条件下重复三次的孔的平均吸光度,并将所得数值处理以使用Graph Pad Prism软件计算EC50,从而提供95%的置信区间。结果如下表1所示。
表1
这些数据显示,本发明的人GDNF变体与成熟野生型人GDNF具有大致相同的与GFRα1受体的结合能力。
实施例3:人GDNF变体和成熟野生型人GDNF均能够诱导RET磷酸化
在来源于小鼠NIH 3T3细胞的MG87RET细胞中进行RET活化和ELISA-RET磷酸化试验,MG87RET细胞稳定转染RET,但不表达GFRα1(参考S.,Fainzilber,M.,Murray-Rust,J.,and />C.F.(1999)EMBO J.18,5901–5910)。MG87RET细胞在无血清Dulbecco改良Eagle培养基中于37℃下饥饿4小时,随后在加或不加100ng/ml GDNF的条件下通过加入1μg/ml人GFRα1(重组人GFRα1-Fc嵌合体,R&D Systems)在37℃下刺激60分钟。用裂解缓冲液(Tris缓冲液、1%Triton X-100、1%Nonidet P-40、10%甘醇、2mM EDTA、1mMNa3VO4、Roche Applied Science的完全Mini蛋白酶抑制剂)裂解后,将裂解物在台式离心机中以10,000rpm离心10min,以沉淀细胞核。将澄清的裂解物加入96孔板(OptiPlate 96FHB,Black;Wallac)中,在4℃下孵育1h,该板先前用0.5μg/ml RET C-20抗体(Santa CruzBiotechnology)包被,并用含2%牛血清白蛋白的Tris缓冲液封闭。使用抗磷酸酪氨酸抗体(4G10;1:1000稀释;Upstate Biotechnology)、抗小鼠辣根过氧化物酶标记抗体(1:3000稀释;DAKO A/S)和增强化学发光反应(Femto ELISA ECL试剂盒;Pierce)检测磷酸化RET。孵育之间的所有洗涤均使用相同的洗涤缓冲液(Tris缓冲液,1%Triton X-100)进行。使用MicroBeta光度计(PerkinElmer Life Sciences)进行信号检测。
结果如图2所示,这些数据显示,本发明的人GDNF变体与成熟野生型人GDNF都能引发RET的磷酸化。
实施例4:人GDNF变体与野生型人GDNF相比更慢地且以更低的水平刺激RET信号传导
用MAPK活化检测***(Gal4-ELK质粒和G4-Luc质粒(参考J.Milbrandt等人,(1998)Neuron 21,1291–1302))和表达人GFRα1的质粒(参考J.Milbrandt等人,同上)转染MG87RET细胞。分别在750g和500g遗传霉素/ml生长培养基存在的条件下选择稳定转染细胞。选择后,在培养基(Dulbecco改良Eagle培养基、10%胎牛血清、normocin、2μg/ml嘌呤霉素、500g/ml遗传霉素、15mM HEPES,pH 7.2)中培养细胞。
测定前6小时,将细胞以400,000个细胞/mL的细胞密度接种在96孔板上。GDNF在无选择性抗生素的生长培养基中稀释至终浓度为100ng/mL,并加入到细胞中保持不同时间。与神经营养因子孵育后,用培养基洗涤细胞一次,并在培养箱中静置24小时以产生荧光素酶,然后加入细胞裂解试剂(Promega)(20μL/孔)。将细胞置于旋转振荡器上孵育15分钟,然后进行一次冻融循环,以确保完全裂解。为了测量荧光素酶活性,向20μl荧光素酶试验试剂(Promega)中加入5μl裂解物,并在MicroBeta光度计(PerkinElmer Life Sciences)上计数。将孵育24小时测定的荧光素酶活性设定为100%,并将不同赋予时间测定的荧光素酶活性标准化至100%,结果如图3所示,这些数据显示,本发明的人GDNF变体虽然仍然能够结合GFRα1并诱导RET磷酸化,但是影响了RET的下游信号传导。与成熟野生型人GDNF相比,本发明的人GDNF变体更慢地且以更低的水平刺激RET信号传导。
实施例5:人GDNF变体和成熟野生型人GDNF在DA转换测试(DA Turnover Assay)中均能够提高多巴胺转换
使用异氟烷(O2中3%)麻醉雄性Sprague-Dawley大鼠。对头部剃毛并用碘溶液消毒,然后将动物放置到带有温度控制垫的立体定位仪上。使用眼用凝胶保护眼部,并使用异氟烷(O2中1-2%)维持麻醉。
在动物头部做正中切口,折回头皮和皮下组织,并将头骨干燥至可见前囟。从前囟和硬脑膜表面测量尾状核的坐标,以用于GDNF输注。将28号输液套管缓慢降低至此位置,并在1分钟后,使用泵开始输注。在4分钟期间以0.5μl/min向左脑半球推注2μl的测试GDNF,并且一旦输注停止,将套管在原位进一步保留3分钟。一旦将套管取出,立即封闭切口位置,施用手术后止痛药,并允许动物在温度控制笼中恢复,然后转移到饲养笼。根据本地伦理准则,在手术后检查动物。在输注后的适当间隔,处死动物,取出脑并准确剖出尾状核,称重并冷冻以待进行多巴胺和代谢物的HPLC分析。
使冷冻组织快速解冻,并在0.5ml的匀浆缓冲剂(0.1M高氯酸(PCA)、0.1mM乙二胺四乙酸(EDTA)、2.5mg/L抗环血酸)中匀浆,此后,在20,000g离心15分钟。取出上清液并过滤通过非注射式过滤装置。使用与电化学检测联用的HPLC进行多巴胺(DA)、二羟基苯乙酸(DOPAC)和高香草酸(HVA)的分析。注入每种样品的20μl等分试样,并相对外部校准曲线(LC4C,BAS,USA)定量。流动相由100mM NaH2PO4、100mM H3PO4、2mM OSA、1mM EDTA、13%甲醇(MeOH)(pH 2.8)组成,在40℃,使用Hypersil BDS(碱钝化硅石)(Thermo Scientific,目录号#28105)150x 3.0mm C183μ颗粒柱。使用Empower色谱软件收集数据。在表示为ng/g湿重组织之前,对所有数据进行4-参数逻辑斯蒂拟合(logistic fit)。多巴胺转换测量值表示为(DOPAC+HVA)/DA,并进行左脑半球(经处理)相比于右脑半球(完好)的比较。
表2
数值为平均值±s.e.m.每组n=5;与完好一侧相比*p<0.001。
这些数据证实了与完好的脑半球相比,人GDNF变体和成熟野生型人GDNF都显著地提高了经处理的脑半球的多巴胺转换。
实施例6:人GDNF变体和成熟野生型人GDNF在体内均能够改善神经病变
6-羟基多巴胺(6-OHDA)-诱导的逆行病变模型(Retrograde Lesion Model)
使用异氟烷(O2中3%)麻醉雄性Sprague-Dawley大鼠。对头部剃毛并用碘溶液消毒,然后将动物放置到带有温度控制垫的立体定位仪上。使用眼用凝胶保护眼部,并使用异氟烷(O2中1-2%)维持麻醉。
在动物头部做正中切口,折回头皮和皮下组织,并将头骨干燥至可见前囟。从前囟和硬脑膜表面测量尾状核的坐标,以用于10μg 6-羟基多巴胺(6-OHDA)的输注。将28号输液套管缓慢降低至此位置,并在1分钟后开始输注。在4分钟期间以0.5μl/min向左脑半球推注2μl的6-OHDA,并且一旦输注停止,将套管在原位进一步保留3分钟。
在6-OHDA输注后30分钟,使用相同的方案输注测试GDNF。GDNF输注的坐标是从上文所述前囟和硬脑膜表面向前后(Anterior-Posterior)+1.0,LM-2.5,DV-4.5mm。
一旦将套管取出,立即封闭切口位置,施用手术后止痛药,并允许动物在温度控制笼中恢复,然后转移到饲养笼。根据本地伦理准则,在手术后检查动物。在输注后的适当间隔,处死动物,取出脑并准确剖出尾状核和黑质,称重并冷冻以待进行多巴胺和代谢物的HPLC分析。
使冷冻组织快速解冻,并在0.5ml的匀浆缓冲剂(0.1M PCA、0.1mM EDTA、2.5mg/L抗环血酸)中匀浆,此后,在20,000xg离心15分钟。取出上清液并过滤通过非注射式过滤装置。使用与电化学检测联用的HPLC进行多巴胺(DA)、DOPAC和HVA的分析。注入每种样品的20μl等分试样,并相对外部校准曲线定量。流动相由100mM NaH2PO4、100mM H3PO4、2mM OSA、1mM EDTA、13%MeOH(pH 2.8)组成,在40℃,使用BDS Hypersil 150x 3.0mm C183μ颗粒柱。使用Empower色谱软件收集数据。在表示为ng/g湿重组织之前,对所有数据进行4-参数逻辑斯蒂拟合。进行左脑半球(经处理)相比于右脑半球(完好)的比较。
表3:尾状核
数值为平均值±s.e.m.每组n=8
与完好一侧相比*p<0.001。
表4:黑质
数值为平均值±s.e.m.每组n=8
与完好一侧相比*p<0.001;与媒介物(处理一侧)相比,#p<0.01。
这些数据显示,向尾状核施用6-OHDA导致经处理一侧的多巴胺水平与完好一侧相比显著降低(表3)。在黑质中也观察到显著的缺陷(表4),其通过施用GDNF而得到预防。通过比较经处理一侧,GDNF变体和野生型GDNF均显著不同于媒介物。
实施例7:人GDNF变体的长期体内给药与成熟野生型人GDNF相比引发显著较低的代偿作用
据报道,GDNF的长期持续过度表达会引起机体的代偿作用,从而导致一系列问题,包括异常出芽、酪氨酸羟化酶下调和体重减轻等。
为了比较人GDNF变体和成熟野生型人GDNF的长期体内给药在代偿作用方面的作用,利用慢病毒载体介导的基因转移技术在大鼠纹状体中过表达GDNF,以研究GDNF对多巴胺能神经元的体内生化效应。
使用四质粒***进行慢病毒载体的生成。将编码成熟野生型人GDNF或人GDNF变体的cDNA克隆在SIN-W-PGK转移载体中。包装构建体使用pCMVΔR-8.92(来源于pCMVΔR-8.91质粒:rev基因编码区中BamHI限制性位点被破坏)。为了进一步降低复制型逆转录病毒的重组和产生风险,将Rev基因***pRSV-Rev质粒中。用pMD.G质粒编码的水泡性口炎病毒G蛋白对病毒颗粒进行假性分型。通过瞬时转染293T细胞产生病毒颗粒。48小时后收集上清液并过滤,采用p24抗原ELISA测定(NenTM Life Science,Boston,MA)测定颗粒含量,超速离心获得高滴度储备液。将沉淀重悬于含1%牛血清白蛋白的磷酸盐缓冲液中,-80℃冷冻储存。
所有病毒载体注射均使用带有33号钝头针头的10μL Hamilton注射器进行立体定向注射。在***/甲苯噻嗪麻醉下,在Sprague-Dawley大鼠每个纹状体的两个不同部位双侧注射2μL的100,000ng p24抗原/mL慢病毒储备液。进行注射的坐标(mm)为(以前囟为参照,根据Paxinos和Watson图谱):(i)AP 1,LAT±3,V-6.2;(ii)AP 0,LAT±3.8,V-6.2。所有坐标的齿条均设置为-2.5mm。注射速度为0.4μL/min。注射后将注射器缩回1mm,并在每个部位再放置4分钟,然后缓慢撤回。对8只大鼠注射lenti-人GDNF变体,并对8只大鼠注射lenti-成熟野生型人GDNF病毒。2只动物未接受病毒载体注射,作为前哨对照。
病毒载体注射后3个月,将lenti-人GDNF变体和lenti-成熟野生型人GDNF组的各4只大鼠以及2只未注射大鼠通过过量戊巴比妥处死,并用磷酸盐缓冲液经心包灌注。使用组织切片机(Stoelting Co,Wood Dale,IL,USA)在中部纹状体水平处获得3mm厚的冠状脑切片。从双侧纹状体中取出直径为1.75mm的打孔组织用于进一步的生化检查。在饱和底物和辅因子条件下(参考Flatmark等人,Eur.J.Biochem.1999,262,840–849),经氧化铝酸(ALOX,Sigma,St Louis,MO,USA)纯化和使用HPLC定量(参考Nagatsu等人,Anal.Biochem.1979,93,82–87)进行TH活性测定。根据Blau等人(J.Inherit.Metab.Dis.1999,22,216–220)所述方法测定组织多巴胺水平。使用学生t检验在每个坐标下比较TH。采用单因素方差分析(ANOVA)和Newman-Keuls事后检验分析酶活性和多巴胺含量测量值。显著性水平设定为p<0.05。数据以平均值±SEM表示。
结果如图4所示,这些数据显示,与未注射组相比,观察到lenti-成熟野生型人GDNF组的酪氨酸羟化酶(TH)活性显著降低(*p<0.05)并且多巴胺水平显著降低(*p<0.05),表明GDNF的长期给药引发了机体的代偿作用。而lenti-人GDNF变体组的TH活性和多巴胺水平虽然也有所变化,但不如野生型组显著,表明本发明的人GDNF变体与野生型GDNF相比不太容易引发机体的代偿作用,因而更适合进行长期给药。
在说明书的描述中,参考术语“一个实施方案”、“具体实施方案”、“实例”等的描述意指结合该实施方案或实例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施方案或实例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施方案或实例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施实施方案或实例中以合适的方式结合。
以上内容仅仅是对本发明所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施方案做各种各样的修改或补充或采用类似的方式替代,只要不偏离发明或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
Claims (10)
1.一种人GDNF变体,其中所述人GDNF变体的氨基酸序列相较于成熟人野生型GDNF的氨基酸序列包含以下氨基酸突变:L48A、I82V、K96P、A100P、L111A和H126R。
2.根据权利要求1所述的人GDNF变体,其包含SEQ ID NO:2的氨基酸序列。
3.根据权利要求1或2所述的人GDNF变体,其与成熟人野生型GDNF相比更慢地且以更低的水平刺激RET信号传导。
4.一种多核苷酸,其包含编码根据权利要求1-3中任一项所述的人GDNF变体的核苷酸序列。
5.一种病毒载体,其包含与根据权利要求4所述的多核苷酸。
6.根据权利要求5所述的病毒载体,其选自重组痘苗病毒、重组腺病毒、重组逆转录病毒、重组腺相关病毒、重组杆状病毒、重组***瘤病毒、重组慢病毒或重组禽痘病毒。
7.一种细胞,其包含根据权利要求4所述的多核苷酸或根据权利要求5或6所述的病毒载体。
8.一种药物组合物,其包含根据权利要求1-3中任一项所述的人GDNF变体、根据权利要求4所述的多核苷酸、根据权利要求5或6所述的病毒载体或根据权利要求7所述的细胞,以及一种或多种药学可接受的稀释剂、载体或赋形剂。
9.根据权利要求8所述的药物组合物,其用于长期给药以治疗帕金森病。
10.根据权利要求1-3中任一项所述的人GDNF变体、根据权利要求4所述的多核苷酸、根据权利要求5或6所述的病毒、根据权利要求7所述的细胞或根据权利要求8或9所述的药物组合物在制备用于长期给药以治疗帕金森病的药物中的用途。
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