CN115894705A - 一种β-NGF融合蛋白、其制备方法及用途 - Google Patents
一种β-NGF融合蛋白、其制备方法及用途 Download PDFInfo
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Abstract
本发明涉及一种β‑NGF融合蛋白、其制备方法及用途。具体地,涉及一种β‑NGF融合蛋白,包含(a)轻链,所述轻链包含β‑NGF;和(b)重链,所述重链包含β‑NGF和Fc片段。本发明通过构建的表达载体和转染宿主细胞可以表达出有生物活性的β‑NGF融合蛋白,制备过程不需要酶切,且获得的融合蛋白含有两个β‑NGF活性分子,活性好,产量高,半衰期长,N端均一。
Description
技术领域
本发明属于生物制药领域,具体地,本发明涉及一种β-NGF融合蛋白分子,本发明还涉及所述β-NGF融合蛋白分子的制备方法及用途。
背景技术
神经生长因子(Nerve Growth Factor,NGF)是人类发现的第一个神经营养因子,首先是由意大利神经科学家Rita Levi-Montalcini和美国生物化学家Stanley Cohen于1956年分离成功;Cohen还意外发现了另一种能促进表皮细胞生长、增殖和分化的生长因子,因而将该因子命名为表皮生长因子(Epidermal Growth Factor,EGF)。为此,Levi-Montalcini和Cohen于1986年共同获得了诺贝尔生理学或医学奖。
神经生长因子(NGF)是神经营养因子中最早被发现,目前研究最为透彻,具有神经元营养和促突起生长双重生物学功能的一种神经细胞生长调节因子,它对中枢及周围神经元发育、分化、生长、再生和功能特性表达均具有重要调控作用。
人NGF基因位于1号染色体短臂2区2带及1号染色体短臂2区1带1号亚带上,由6个外显子及5个内含子组成。
NGF最初是在3种蛋白质亚基组成的7S、130kDa复合物中,该复合物由α-NGF、β-NGF和γ-NGF(2:1:2比例,图1)三个蛋白组成。这种形式的NGF也称为proNGF(NGF前体)。该复合物的α亚单位属于非匀质性酸蛋白,其pI约为4.3,它的作用主要是阻止γ亚单位对β亚单位的水解,从而对其起到保护作用。γ亚单位具有精氨酸酯肽酶活性,其主要作用与NGF的合成有关,可以将无活性的β亚基激活。
我们说的神经生长因子通常是指蛋白质的2.5S、26kDa的β亚基(即β-NGF),是生物活性的7S NGF复合物的唯一组分(即充当信号分子)。β-NGF,它是由两条含有118个氨基酸的肽链通过非共价键形成的二聚体。成熟的NGF链内含有6个半胱氨酸残基,可以产生3对链内二硫键,二硫键对维持NGF的生物学活性十分重要,一旦二硫键被破坏,NGF的生物学活性会大大降低甚至是丧失。
NGF是一种参与神经元分化和维护的内源性蛋白质,这种内源性蛋白质通过多种机制为眼角膜完整性带来有力的支持。NGF直接作用于角膜细胞来刺激细胞的生长与存活。此外,已知NGF能结合泪腺上的受体(图2),促进泪液分泌,为眼睛提供润滑和自然保护,免受病原体伤害。这种蛋白质经证实能够支持角膜神经支配,即神经营养性角膜炎当中已经损失的一种神经功能。
OxervateTM(cenegermin,重组人神经生长因子,rhNGF)是意大利制药商Dompé原研的一款滴眼液,是一种用于中度至重度神经营养性角膜炎(neurotrophic keratitis,NK)成人患者的治疗的孤儿药,目前已在欧盟、美国和中国上市。另外,其用于治疗干眼病的适应症在研,二期临床结果良好。但是,每2小时滴眼一次,使用很不方便。
鼠神经生长因子已经在中国上市多年,用于治疗外周神经损伤。T1/2(β)为4.83小时,每天注射一次。但鼠神经生长因子有10%氨基酸与人的不一样,有潜在免疫原性的风险。
为了制备重组人神经生长因子,原研厂商首先表达proNGF包涵体,然后再变复性,纯化proNGF复性液,酶切纯化得到重组人神经生长因子。然而,该方法需要酶切,酶切后还需要纯化,步骤较多,过程较为繁琐。这种方法制备的NGF热源不容易去除,半衰期短。
中国专利CN108300736B提供了一种CHO细胞制备rhNGF-FC的方法,但其一个rhNGF-FC分子只含有一个NGF活性分子,表达量为18mg/L,况且N端不均一,需要furin酶切。
鉴于现有技术中存在的上述问题,本申请的发明人拟开发一种活性好,产量高,半衰期长,N端均一的β-NGF融合蛋白。
发明内容
针对现有技术的不足,本发明提供了一种β-NGF融合蛋白分子。该β-NGF融合蛋白分子含有两个β-NGF活性分子,活性好,产量高,半衰期长,N端均一。
因此,本发明的第一个目的在于提供一种β-NGF融合蛋白,包含:
(a)轻链,所述轻链包含β-NGF;和
(b)重链,所述重链包含β-NGF和Fc片段。
本发明第二个目的在于提供编码所述β-NGF融合蛋白的分离的核酸分子。
本发明的第三个目的在于提供含有所述核酸分子的表达载体。
本发明的第四个目的在于提供含有所述表达载体的宿主细胞。
本发明第五个目的在于提供所述β-NGF融合蛋白的制备方法。
本发明第六个目的在于提供含有所述β-NGF融合蛋白的药物组合物。
本发明第七个目的在于提供所述β-NGF融合蛋白或所述药物组合物与另外的治疗剂使用。
本发明的第八个目的在于提供所述β-NGF融合蛋白或所述药物组合物的用途。
为了实现上述目的,本发明采用了如下技术方案:
在本发明的第一方面,提供一种β-NGF融合蛋白,包含:
(a)轻链,所述轻链包含β-NGF;和
(b)重链,所述重链包含β-NGF和Fc片段。
在另一优选例中,所述β-NGF为人β-NGF。
在另一优选例中,所述β-NGF包含如SEQ ID NO:1所示的氨基酸序列。
在另一优选例中,所述β-NGF和所述Fc片段通过连接子连接。
在另一优选例中,所述重链从N端到C端依次为β-NGF、连接子和Fc片段。
在另一优选例中,所述连接子包含通式(GGGGS)n,n为整数,n=1~10;较佳地n=4~10;优选地n=4,氨基酸序列如SEQ ID NO:3所示。
在另一优选例中,所述Fc片段来源于IgG。
在另一优选例中,所述IgG来源于哺乳动物;较佳地来源于鼠、食蟹猴或人;优选来源于人。
在另一优选例中,所述Fc片段选自IgG1、IgG2、IgG3或IgG4;较佳地选自IgG4。
在另一优选例中,所述Fc片段包含人IgG铰链区、CH2和CH3区。
在另一优选例中,所述Fc片段包含人IgG4铰链区、CH2和CH3区。
在另一优选例中,所述Fc片段包含如SEQ ID NO:2所示氨基酸序列。
在另一优选例中,所述Fc片段包含Fc变体,所述Fc变体包含一个或多个氨基酸的取代、缺失和/或插入。
在另一优选例中,所述重链包含如SEQ ID NO:4所示的氨基酸序列。
在另一优选例中,所述轻链包含如SEQ ID NO:1所示的氨基酸序列。
在另一优选例中,所述β-NGF融合蛋白包含如SEQ ID NO:4和/或SEQ ID NO:1所示的氨基酸序列。
在本发明的第二方面,提供了一种分离的核酸分子,所述核酸分子包含编码如本发明的第一方面所述的β-NGF融合蛋白的核苷酸序列。
在另一优选例中,所述核酸分子包含如SEQ ID NO:8所示轻链核苷酸序列和如SEQID NO:9所示重链核苷酸序列。
在另一优选例中,所述核酸分子包含如SEQ ID NO:11所示轻链核苷酸序列和如SEQ ID NO:12所示重链核苷酸序列。
在另一优选例中,所述核酸分子包含如SEQ ID NO:15所示轻链核苷酸序列和如SEQ ID NO:16所示重链核苷酸序列。
在本发明的第三方面,提供了一种表达载体,所述的表达载体包含本发明的第二方面所述的核酸分子。
在另一优选例中,所述表达载体为选自pCGS3、pcDNA3.4、pDR1、pcDNA3.1(+)、pcDNA3.1/ZEO(+)、pDHFR或pTT5;较佳地选自pCGS3。
在本发明的第四方面,提供了一种宿主细胞,所述宿主细胞包含本发明的第三方面所述的表达载体。
在另一优选例中,所述宿主细胞为选自COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、CHO-K1、293E或Expi293F细胞;较佳地选自CHO细胞、293E或Expi293F细胞;更佳地为CHO细胞。
在本发明的第五方面,提供了一种β-NGF融合蛋白的制备方法,其特征在于,所述方法包括:
(a)在表达条件下,培养上述的宿主细胞,从而表达β-NGF融合蛋白;
(b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
在本发明的第六方面,提供了一种药物组合物,所述药物组合物包含如本发明的第一方面所述的β-NGF融合蛋白和药学上可接受的载体。
在本发明的第七方面,提供了如本发明的第一方面所述的β-NGF融合蛋白或本发明第六方面所述的药物组合物,其与另外的治疗剂分别、依次或同时使用。
在本发明的第八方面,提供了一种如本发明的第一方面所述的β-NGF融合蛋白或如本发明的第六方面所述的药物组合物用于制备治疗中度至重度神经营养性角膜炎或者外周神经损伤药物的用途。
本发明的第九方面,提供了一种治疗疾病的方法,包括步骤:给需要的对象施用如本发明的第一方面所述的β-NGF融合蛋白,或如本发明的第六方面所述的药物组合物。
本发明的有益效果:本发明提供了一种β-NGF融合蛋白分子,该β-NGF融合蛋白分子含有两个β-NGF活性分子,活性好,产量高,半衰期长,N端均一。
附图说明
图1表示NGF结构。
图2表示NGF与NGF受体(NGFR)结合示意图。
图3表示β-NGF融合蛋白结构。
图4表示分离纯化获得的β-NGF融合蛋白SDS-PAGE鉴定图。
图5表示ELISA法检测β-NGF融合蛋白体外生物活性。
图6表示TF-1细胞法检测β-NGF融合蛋白体外生物活性。
图7A-7C表示β-NGF融合蛋白质谱检测图,图7B-7C为图7A的局部放大图。
具体实施方式
本发明中,术语“融合蛋白”指一种β-NGF融合蛋白,包含(a)轻链,所述轻链包含β-NGF;和(b)重链,所述重链包含β-NGF和Fc片段。
应理解,本发明还提供一种β-NGF融合蛋白的制备方法,即构建本发明β-NGF融合蛋白的方法。所述方法如本发明第五方面所述。通过本发明的构建方法获得的β-NGF融合蛋白,含有两个β-NGF活性分子,活性好,产量高,半衰期长,N端均一。
本发明中,术语“Fc片段”和“免疫球蛋白Fc片段”可以互换使用,均指含有免疫球蛋白铰链区、免疫球蛋白重链恒定区2(CH2)和重链恒定区3(CH3)且不含免疫球蛋白重链可变区和轻链可变区、重链恒定区1(CH1)和轻链恒定区(CL)的蛋白质。此外,除了重链可变区和轻链可变区之外,本发明的免疫球蛋白Fc片段可能包含重链恒定区1(CH1)和/或轻链恒定区(CL)的一部分或全部。同样,只要它具有与天然蛋白质基本上相似或优于天然蛋白质的生理学功能,IgG Fc片段就可以是在CH2和/或CH3氨基酸序列的相对较长部分中有缺失的片段。即,本发明的免疫球蛋白Fc片段可能包含1)CH1结构域、CH2结构域、CH3结构域和CH4结构域,2)CH1结构域和CH2结构域,3)CH1结构域和CH3结构域,4)CH2结构域和CH3结构域,5)一个或多个结构域和免疫球蛋白铰链区(或铰链区的一部分)的组合,或6)重链恒定区和轻链恒定区各个结构域的二聚体。
本发明的Fc片段包括天然氨基酸序列和其变体。氨基酸序列衍生物是由于一个或多个氨基酸残基的缺失、插入、非保守性或保守性取代或它们的联合而不同于天然氨基酸序列的序列。
此外,如果需要,Fc片段可以通过磷酸化、硫酸化、丙烯酸化、糖基化、甲基化、法呢基化、乙酰化、酰胺化等等作用被修饰。
上述Fc衍生物是具有与本发明Fc片段同样的生物学活性或提高了结构稳定性(例如抗热、pH等等)的衍生物。
此外,这些Fc片段可获自从人和包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠在内的其它动物中分离的天然形式,或可以是重组体或它们的衍生物,获自被转化的动物细胞或微生物。在此,它们可通过从人或动物体中分离完整的免疫球蛋白并用蛋白水解酶加以处理而从天然免疫球蛋白获得。木瓜蛋白酶将天然的免疫球蛋白消化成Fab和Fc片段,而胃蛋白酶处理则导致了pF′c和F(ab′)2片段的产生。可对这些片段进行例如大小排阻层析,以分离Fc或pF′c。
优选的,人源Fc片段是获自微生物的重组免疫球蛋白Fc片段。
此外,本发明的免疫球蛋白Fc片段可以是具有天然糖链、与天然形式相比糖链增加或与天然形式相比糖链减少的形式,或者可以是去糖基化的形式。免疫球蛋白Fc糖链的增加、减少或去除可以通过本领域的通用方法完成,诸如化学法、酶促法和利用微生物的遗传工程方法。
另一方面,免疫球蛋白Fc片段可来自人类或其它动物,包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠,优选人类。此外,免疫球蛋白Fc片段可以是来自IgG、IgA、IgD、IgE和IgM的Fc片段,或者通过它们的组合或杂合制备而来的Fc片段。优选Fc片段来自IgG或IgM,它们是人类血液中最丰富的蛋白质之一,最优选来自IgG(已知它能延长配体结合蛋白质的半衰期)。
本发明中,术语“连接子”是指β-NGF和Fc片段之间的短的连接子序列,优选为柔性接头。合适的连接子实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基,接头中氨基酸残基的标识和序列可随着连接子中需要实现的次级结构要素的类型而变化。
本发明提供了一种核酸分子,所述核酸分子编码上述的轻链。其中将β-NGF基因,通过加入前体pro序列、信号肽signal 1序列、Kozak序列以及密码子优化后获得了轻链基因和相关元件序列(SEQ ID NO:8)。
本发明还提供了一种核酸分子,所述核酸分子编码上述的重链(β-NGF+(G4S)4+Fc)。其中将β-NGF+(G4S)4+Fc基因,通过加入前体pro序列、信号肽signal 2序列、Kozak序列以及密码子优化后获得了重链基因和相关元件序列(SEQ ID NO:9)。
本发明核酸分子的制备方法为本领域常规的制备方法,较佳地包括以下制备方法:通过基因克隆技术例如PCR方法等,获得编码上述多肽的核酸分子,或者通过人工全序列合成的方法得到编码上述多肽的核酸分子。
本发明中,术语“表达载体”含有上述任一所述的核酸分子,为本领域常规的表达载体,是指包含适当的调控序列,例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒,如适当的噬菌体或者噬菌粒,更多技术细节请参见例如Sambrook等,MolecularCloning:A Laboratory Manual,第二版,Cold Spring Harbor Laboratory Press,1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology,第二版,Ausubel等编著。
本发明所述表达载体较佳地为选自pCGS3,pcDNA3.4,pDR1,pcDNA3.1(+),pcDNA3.1/ZEO(+),pDHFR和pTT5,优选自pCGS3。
本发明中,术语“宿主细胞”含有上述表达载体,为本领域常规的各种宿主细胞,只要能满足使上述重组表达载体稳定地自行复制,且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞,较佳地包括:COS、CHO(中国仓鼠卵巢,Chinese Hamster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1,更佳地为E.coliTG1、BL21(DE3)细胞或者CHO-K1细胞。将前述表达载体转化至宿主细胞中,即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法,较佳地为化学转化法,热激法或电转法。
作为优选的方案,本发明所述宿主细胞是真核细胞,优选自CHO细胞,293E和Expi293F细胞。
本发明提供了一种制备方法,所述制备方法包括以下步骤:
(a)在表达条件下,培养上述的宿主细胞,从而表达β-NGF融合蛋白;
(b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
更具体地,用SEQ ID NO:8编码轻链的核苷酸分子和用SEQ ID NO:9编码重链的核苷酸分子分别插入pCGS3不同的多克隆位点,构建表达载体;在CHO细胞中分泌表达;离心分离收集上清液,用Protein A亲和层析纯化,得到高纯度的β-NGF融合蛋白。
本发明所述的宿主细胞的培养方法、所述β-NGF融合蛋白的分离和纯化方法为本领域常规方法,具体操作方法请参考相应的细胞培养技术手册以及蛋白分离纯化技术手册。利用上述方法,可以将β-NGF融合蛋白纯化为基本均一的物质,例如在非还原SDS-PAGE电泳上为单一条带。
本发明所述纯化后β-NGF融合蛋白分子具有生物活性,可以作为治疗疾病的药物。
本发明中,术语“药物组合物”是指本发明的β-NGF融合蛋白和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效,这些制剂可以保证本发明公开的β-NGF融合蛋白的氨基酸核心序列的构象完整性,同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。有效量的本发明的β-NGF融合蛋白或其药物组合物施用受试者后,在治疗的个体中产生预期效果的量或剂量,该预期效果包括个体病症的改善。其中受试者包括但不限于哺乳动物,例如人、非人灵长类动物、大鼠和小鼠等。此外,本发明的β-NGF融合蛋白或其药物组合物还可与其他治疗剂一起使用。
以下实施例是对本发明进行进一步的说明,不应理解为是对本发明的任何限制。实施例不包括对传统方法的详细描述,如那些将已知氨基酸序列表达纯化获得多肽片段的方法,用于构建载体和质粒的方法,将编码蛋白的基因插入到载体和质粒的方法或将质粒引入宿主细胞的方法等。这样的方法对于本领域中具有普通技术的人员是众所周知的,并且在许多出版物中都有所描述,包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual 2nd edition,Cold spring HarborLaboratory Press.
以下实施例中使用的检测方法说明如下:
1、SDS-PAGE检测:
检测系统:Mini protein Tetra system
检测条件:140V恒压45-55min
2、紫外检测:
仪器型号:Nanodrop one(Thermo)
消光系数:1.42
3、Protein A亲和层析
层析柱:XK16/20(GE)
填料:MabSelectTMSuReTM(GE)
层析系统:AKTA Pure150(GE)
操作系统:unicorn 7.0(GE)
流速;1.0mL/min
4、ELISA检测及处理
酶标仪:SpectraMax 190,波长450nm
处理软件:GraphPad Prism 9
以下实施例中使用的实验材料和实验试剂,如果没有特殊说明,均为常规商购获得。
实施例1.β-NGF融合蛋白表达载体的构建
1.β-NGF融合蛋白氨基酸的设计,以及其基因和相关元件序列的设计
将轻链和重链用不同的表达框分别表达。编码轻链中β-NGF的核苷酸序列如SEQID NO:15所示,编码重链中β-NGF+Fc的核苷酸序列如SEQ ID NO:16所示。为了便于表达折叠,分别在轻链和重链β-NGF的N端增加前体序列Pro(SEQ ID NO:5);为了使融合蛋白分泌到发酵液上清中,在轻链和重链NGF前体序列Pro的N端分别增加信号肽signal 1(SEQ IDNO:6)和信号肽signal 2(SEQ ID NO:7)。编码轻链signal 1+ProNGF氨基酸的核苷酸序列用CHO偏好的密码子优化,得到优化后的核苷酸序列(SEQ ID NO:8);编码重链signal 2+ProNGF+Fc氨基酸的核苷酸序列用CHO偏好的密码子优化,得到优化后的核苷酸序列(SEQID NO:9)。为了提高表达量,在编码轻链signal 1+ProNGF氨基酸的核苷酸(SEQ ID NO:8)5’端添加Kozak序列:GCCACC(SEQ ID NO:10),为了便于克隆,在GCCACC(SEQ ID NO:10)5’端添加内切酶HindIII位点,在SEQ ID NO:8的3’端添加内切酶Xhol位点得到轻链基因signal 1+ProNGF和相关元件序列(SEQ ID NO:11)。同理为了提高表达量,在编码重链signal 2+ProNGF+Fc氨基酸的核苷酸(SEQ ID NO:9)5’端添加Kozak序列:GCCACC(SEQ IDNO:10),为了便于克隆,在GCCACC(SEQ ID NO:10)5’端添加内切酶BstB I位点,在SEQ IDNO:9的3’端添加内切酶Pac I位点得到重链基因signal 2+ProNGF+Fc和相关元件序列(SEQID NO:12)。
2.β-NGF融合蛋白表达载体的制备
全基因合成重链基因signal 2+ProNGF+Fc和相关元件序列(SEQ ID NO:12)和轻链基因signal 1+ProNGF和相关元件序列(SEQ ID NO:11),分别插入pCGS3不同的表达框,构建β-NGF Fusion-pCGS3和质粒菌。大量提取质粒β-NGF Fusion-pCGS3,用内切酶Pvu1(NEB公司)线性化,0.22μm无菌过滤除菌备用。
实施例2.β-NGF融合蛋白CHO稳转株的构建
用线性化的质粒β-NGF Fusion-pCGS3转染CHO细胞,先对细胞池进行加压筛选,随后挑取单克隆,最后针对每个单克隆进行加压扩大培养继续筛选。用Protein A探针测β-NGF融合蛋白的表达量,挑选表达量最高的细胞株进行后续试验,表达量最高为1.164g/L。
实施例3.β-NGF融合蛋白表达纯化
1.β-NGF融合蛋白表达
将实施例2中获得的β-NGF融合蛋白重组细胞株进行流加培养。将表达最高的细胞株从冻存中复苏,每3-4天传代。细胞从生长活力恢复后,将细胞接种至Gibco ExpiCHO培养基中。最少经过3次传代,直到细胞完全地适应培养基。细胞在培养过程中倍增时间小于24小时可默认为已完全适应。在完全适应后,随后将细胞以0.5×106/mL接种至30mL体积的摇瓶中。培养的第3天开始流加补料。培养过程中使用250g/L的葡萄糖溶液将葡萄糖控制在3g/L以上。
2.Protein A亲和层析分离纯化β-NGF融合蛋白
使用Purifier150层析系统来进行亲和层析,色谱柱为MabSelectTMSuReTM1mL。仪器操作按操作指南进行,A1泵为Buffer A(PBS,pH7.4),B1泵为Buffer B(50mM柠檬酸缓冲液,pH2.5)。
清洗:以0.1M NaOH,流速1mL/min,冲洗Protein A凝胶柱5个柱体积(CV),再用超纯水以流速1mL/min清洗5CV,去除Protein A凝胶上的杂质残留。
平衡:Buffer A以1mL/min流速冲洗10CV,以维持Protein A凝胶环境适合β-NGF融合蛋白与Protein A的结合。
上样:澄清过滤后的发酵液以1mL/min的速率通过Protein A凝胶,使β-NGF融合蛋白与Protein A能特异性的结合。
冲平:Buffer A以1mL/min流速冲洗至280nm吸收值低于0.01。
洗脱:以1mL/min流速,Buffer B冲洗Protein A凝胶,收集洗脱峰。
中和:洗脱峰收集样品用1.5mol/L Tris-HCl,pH9.0调pH至5.6。SDS-PAGE检测分析纯化得到的β-NGF融合蛋白。其结构示意图如图3所示,SDS-PAGE如图4所示。
实施例4.β-NGF融合蛋白ELISA检测
1、包被抗原β-NGF(NGF,SEQ ID NO:1),每孔100μL,浓度为80nM,,于4℃孵育过夜;包被β-NGF融合蛋白(β-NGF Fusion),每孔100μL,浓度为80nM,于4℃孵育过夜。
2、洗涤:取出包被好抗原的Elisa板,用洗涤缓冲液PBST洗板3次,拍干Elisa板等待下一步封闭。
3、封闭:每孔加入200μL封闭液(PBST+1%BSA),于室温封闭2h。
4、洗涤:用洗涤缓冲液PBST洗板3次,拍干备用。
5、加入一抗Fulranumab(轻链氨基酸序列如SEQ ID NO:13所示,重链氨基酸序列如SEQ ID NO:14所示;参考IMGT数据库)
(1)稀释抗体:在96孔细胞培养板中进行一抗稀释(稀释液为封闭液):初始浓度为1μg/mL,从左至右依次以2倍稀释,稀释10个梯度,第11和12列设置为不加一抗的空白对照。每个样品,每个梯度设置2个复孔。
(2)每孔加入100μL稀释好的一抗,于室温封闭2h。
6、洗涤:用洗涤缓冲液PBST洗板3次,拍干备用。
7、加入二抗:按1:1000的比例用封闭液(PBST+1%BSA)稀释Fab HRP(苏州博特龙),每孔加入100μL稀释好的二抗,于室温避光孵育1h。
8、洗涤:用洗涤缓冲液PBST洗板3次,拍干备用。
9、显色:每孔加入100μL新鲜配制的显色液(TMB),于室温避光孵育10分钟。
10、终止:每孔加入70μL终止液(2M H2SO4),混匀后,立即到酶标仪上读数,检测波长为450nm。
处理数据图谱如图5所示。抗原β-NGF的EC50为0.534nM,β-NGF融合蛋白的EC50为0.524nM,说明β-NGF融合蛋白具有生物活性。
实施例5.TF-1细胞法体外生物活性分析
TF-1细胞法是基于TF-l细胞系的粒细胞巨噬细胞集落刺激因子(GM-CSF)高度依赖性利用NGF与TF-1细胞表面的NGF高亲和力受体TrkA结合后诱导TF-1细胞增殖的特点,建立的一种新的NGF活性测定方法。该方法目前已成为检测重组人神经生长因子(rhNGF)WHO参考品的标准方法。
材料:1640培养基,Gibco,货号:22400-105;96孔平底细胞培养板,Corning,货号CLS3599;CCK-8试剂盒,Dojindo,货号CK04;胎牛血清,GIBCO,货号10099-141;TF-1细胞,ATCC,CRL-2003TM
1.处于对数生长期的TF-1细胞用37℃预热的1640培养基洗2遍,300-500g离心5分钟;
2.计数TF-1细胞,用含10%FBS的1640培养基悬浮到适当密度,接种到96孔板中,10000个/150μL/孔;
3.在96孔板中用1640培养基梯度稀释β-NGF(NGF)样品或者β-NGF融合蛋白(β-NGFFusion)样品,100nM起始,以三倍连续稀释9个梯度;将稀释好的样品加入96孔细胞培养板中,50μL/孔;96孔周围用200μL/孔的蒸馏水补齐;
4.在37℃、5%CO2条件的孵箱中孵育培养3天;
5.在96孔细胞培养板每孔加入20μL CCK-8溶液,在37℃孵箱中继续培养8h;
6.混匀后酶标仪读OD450值,GraphPad Prism6进行数据分析,作图并计算EC50。
生物活性如图6所示,抗原β-NGF的EC50为0.045nM,β-NGF融合蛋白的EC50为0.292nM,由此可知,β-NGF融合蛋白具有生物活性。
实施例6.质谱检测
质谱条件如下:
Waters公司UPLC-XEVO G2 Q-TOF液质联用系统。系统液相部分配置为:BSM二元高压混合泵,SM样品管理器,TUV紫外检测器;质谱部分配置为:ESI源,Q-TOF检测器。数据处理分析采用Masslynx V4.1及BiopharmaLynx分析软件(Version:1.2)。
液相条件
色谱柱:Mass PREPTM Micro Desalting Column 2.1×5mm(完整蛋白分子量分析),柱温:80℃;
流动相A:0.1%FA-H2O
流动相B:0.1%FA-CAN
Seal Wash溶液:10%IPA
质谱清洗液:50%ACN
质谱IntelliStart阀清洗液:50%MeOH
进样体积:10μL
样品室温度:10℃
梯度洗脱条件:
表1.梯度洗脱条件
质谱条件
MS数据均采用轮廓图(continuum)模式,在Resolution模式下采集;LockSpray
采集模式为:实时采集并不应用校准。
校准溶液:实时校准(LockSpray)溶液:2ng/μL LE溶液;
质量轴的校正溶液:2μg/μL碘化钠溶液。
质谱参数:
表2.质谱参数
脱N糖分子量:N-糖酰胺酶(PNGase)切除连接在天冬酰胺残基(Asn)上的糖链,再进行质谱检测。
表3.β-NGF融合蛋白脱N-糖完整分子量
检测结果如图7A-7C和表3所示,由此可知,本发明的β-NGF融合蛋白N端均一,与设计完全一致。
序列表
SEQ ID NO:1(轻链氨基酸序列)
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR
SEQ ID NO:2(Fc氨基酸序列)
AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO:3(Linker氨基酸序列)
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO:4(重链氨基酸序列)
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRGGGGSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO:5(NGF前体Pro氨基酸序列)
EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKR
SEQ ID NO:6(signal 1氨基酸序列)
MDTRAPTQLLGLLLLWLPGARC
SEQ ID NO:7(signal 2氨基酸序列)
METGLRWLLLVAVLKGVQC
SEQ ID NO:8(轻链核苷酸序列)
ATGGACACTAGAGCCCCCACCCAGCTGCTGGGCCTCTTGCTTCTCTGGCTGCCGGGGGCGCGCTGTGAGCCCCACAGTGAGAGCAATGTACCTGCCGGGCACACCATTCCACAGGCCCACTGGACTAAACTGCAGCACTCCCTGGATACAGCCTTGCGGCGCGCCCGCTCCGCTCCTGCTGCTGCAATAGCAGCGCGGGTGGCCGGCCAGACCAGGAACATAACTGTGGACCCTCGTTTATTTAAGAAGCGCAGACTCAGATCTCCAAGGGTTCTTTTCTCAACCCAACCACCAAGAGAAGCAGCTGACACACAGGACCTGGACTTTGAGGTGGGCGGTGCCGCGCCCTTCAACAGGACACATAGGAGCAAACGCTCTTCCAGCCATCCGATCTTCCATCGGGGAGAGTTCAGTGTCTGTGATAGCGTCTCAGTATGGGTGGGAGATAAGACAACAGCAACAGACATCAAAGGGAAAGAAGTCATGGTGCTAGGAGAGGTGAATATCAACAACTCTGTTTTCAAGCAGTATTTTTTTGAAACCAAGTGCAGAGATCCCAATCCTGTGGATTCGGGCTGCCGAGGTATCGACTCCAAGCACTGGAATAGCTACTGCACGACGACTCACACTTTTGTGAAAGCACTCACCATGGATGGAAAGCAAGCTGCCTGGAGGTTCATTCGGATTGACACCGCCTGTGTCTGCGTGCTGAGTCGGAAAGCTGTTCGATGA
SEQ ID NO:9(重链核苷酸序列)
ATGGAGACTGGGCTGAGGTGGCTGTTACTAGTTGCTGTCCTGAAGGGGGTCCAGTGTGAGCCCCACAGCGAAAGCAATGTGCCGGCAGGACACACCATACCTCAGGCCCACTGGACAAAGCTGCAGCATTCTTTGGACACTGCTCTTCGCAGAGCCCGGTCTGCACCTGCCGCTGCCATCGCAGCCAGGGTGGCCGGACAGACTAGAAACATTACGGTTGACCCCCGGCTCTTCAAGAAGCGTCGCCTTCGCAGCCCCCGGGTGCTGTTTTCAACTCAGCCCCCCAGGGAGGCGGCTGATACCCAAGATCTGGACTTTGAGGTTGGAGGAGCTGCGCCTTTTAACAGGACCCACAGGTCCAAGCGATCTAGTTCTCATCCCATCTTCCACCGGGGGGAATTTAGTGTGTGTGACTCCGTGTCAGTGTGGGTGGGAGACAAGACCACCGCAACTGACATCAAGGGCAAGGAGGTGATGGTGCTGGGAGAGGTGAACATCAATAATTCAGTTTTCAAACAGTACTTCTTTGAAACAAAGTGTAGAGATCCGAACCCTGTTGATAGCGGCTGCAGGGGCATTGACAGCAAGCACTGGAACAGCTACTGCACCACGACACACACATTTGTGAAAGCGCTCACCATGGATGGGAAGCAGGCAGCCTGGCGCTTCATCCGGATTGATACAGCCTGCGTCTGTGTCCTAAGTAGAAAAGCTGTTCGAGGTGGCGGGGGCAGTGGCGGCGGCGGATCTGGAGGGGGTGGTTCCGGCGGGGGCGGCTCTGCCGAGAGCAAATATGGTCCTCCCTGCCCGCCGTGCCCAGCACCAGAAGCAGCTGGAGGTCCATCGGTGTTTCTCTTCCCACCCAAACCTAAAGATACACTGATGATCTCCCGGACACCAGAGGTCACTTGTGTGGTCGTGGATGTCTCCCAGGAAGACCCCGAGGTACAGTTCAACTGGTACGTGGACGGAGTGGAAGTGCACAACGCCAAAACCAAGCCTAGAGAAGAGCAGTTTAATAGCACGTATCGTGTAGTGTCCGTATTAACAGTCCTCCATCAAGACTGGCTCAACGGGAAAGAATATAAATGCAAGGTGAGCAACAAAGGACTCCCTTCTTCCATTGAGAAGACAATATCTAAGGCCAAGGGTCAGCCAAGAGAACCCCAAGTCTATACTCTGCCCCCGTCACAAGAAGAGATGACCAAAAATCAGGTAAGTCTTACCTGCCTGGTAAAGGGTTTCTACCCCAGTGATATTGCCGTAGAGTGGGAATCTAATGGCCAGCCTGAAAATAACTACAAGACTACCCCACCAGTGCTTGACTCGGATGGGAGCTTCTTCCTGTACTCCCGCCTGACGGTTGACAAATCACGATGGCAGGAGGGCAATGTCTTCAGCTGTTCGGTCATGCACGAGGCTTTGCATAATCATTATACGCAAAAGTCCCTTTCCTTGAGTCTGGGCTGA
SEQ ID NO:10(Kozak核苷酸序列)
GCCACC
SEQ ID NO:11(signal 1+ProNGF和相关元件核苷酸序列)AAGCTTGCCACCATGGACACTAGAGCCCCCACCCAGCTGCTGGGCCTCTTGCTTCTCTGGCTGCCGGGGGCGCGCTGTGAGCCCCACAGTGAGAGCAATGTACCTGCCGGGCACACCATTCCACAGGCCCACTGGACTAAACTGCAGCACTCCCTGGATACAGCCTTGCGGCGCGCCCGCTCCGCTCCTGCTGCTGCAATAGCAGCGCGGGTGGCCGGCCAGACCAGGAACATAACTGTGGACCCTCGTTTATTTAAGAAGCGCAGACTCAGATCTCCAAGGGTTCTTTTCTCAACCCAACCACCAAGAGAAGCAGCTGACACACAGGACCTGGACTTTGAGGTGGGCGGTGCCGCGCCCTTCAACAGGACACATAGGAGCAAACGCTCTTCCAGCCATCCGATCTTCCATCGGGGAGAGTTCAGTGTCTGTGATAGCGTCTCAGTATGGGTGGGAGATAAGACAACAGCAACAGACATCAAAGGGAAAGAAGTCATGGTGCTAGGAGAGGTGAATATCAACAACTCTGTTTTCAAGCAGTATTTTTTTGAAACCAAGTGCAGAGATCCCAATCCTGTGGATTCGGGCTGCCGAGGTATCGACTCCAAGCACTGGAATAGCTACTGCACGACGACTCACACTTTTGTGAAAGCACTCACCATGGATGGAAAGCAAGCTGCCTGGAGGTTCATTCGGATTGACACCGCCTGTGTCTGCGTGCTGAGTCGGAAAGCTGTTCGATGACTCGAG
SEQ ID NO:12(signal 2+ProNGF+Fc和相关元件核苷酸序列)
TTCGAAGCCACCATGGAGACTGGGCTGAGGTGGCTGTTACTAGTTGCTGTCCTGAAGGGGGTCCAGTGTGAGCCCCACAGCGAAAGCAATGTGCCGGCAGGACACACCATACCTCAGGCCCACTGGACAAAGCTGCAGCATTCTTTGGACACTGCTCTTCGCAGAGCCCGGTCTGCACCTGCCGCTGCCATCGCAGCCAGGGTGGCCGGACAGACTAGAAACATTACGGTTGACCCCCGGCTCTTCAAGAAGCGTCGCCTTCGCAGCCCCCGGGTGCTGTTTTCAACTCAGCCCCCCAGGGAGGCGGCTGATACCCAAGATCTGGACTTTGAGGTTGGAGGAGCTGCGCCTTTTAACAGGACCCACAGGTCCAAGCGATCTAGTTCTCATCCCATCTTCCACCGGGGGGAATTTAGTGTGTGTGACTCCGTGTCAGTGTGGGTGGGAGACAAGACCACCGCAACTGACATCAAGGGCAAGGAGGTGATGGTGCTGGGAGAGGTGAACATCAATAATTCAGTTTTCAAACAGTACTTCTTTGAAACAAAGTGTAGAGATCCGAACCCTGTTGATAGCGGCTGCAGGGGCATTGACAGCAAGCACTGGAACAGCTACTGCACCACGACACACACATTTGTGAAAGCGCTCACCATGGATGGGAAGCAGGCAGCCTGGCGCTTCATCCGGATTGATACAGCCTGCGTCTGTGTCCTAAGTAGAAAAGCTGTTCGAGGTGGCGGGGGCAGTGGCGGCGGCGGATCTGGAGGGGGTGGTTCCGGCGGGGGCGGCTCTGCCGAGAGCAAATATGGTCCTCCCTGCCCGCCGTGCCCAGCACCAGAAGCAGCTGGAGGTCCATCGGTGTTTCTCTTCCCACCCAAACCTAAAGATACACTGATGATCTCCCGGACACCAGAGGTCACTTGTGTGGTCGTGGATGTCTCCCAGGAAGACCCCGAGGTACAGTTCAACTGGTACGTGGACGGAGTGGAAGTGCACAACGCCAAAACCAAGCCTAGAGAAGAGCAGTTTAATAGCACGTATCGTGTAGTGTCCGTATTAACAGTCCTCCATCAAGACTGGCTCAACGGGAAAGAATATAAATGCAAGGTGAGCAACAAAGGACTCCCTTCTTCCATTGAGAAGACAATATCTAAGGCCAAGGGTCAGCCAAGAGAACCCCAAGTCTATACTCTGCCCCCGTCACAAGAAGAGATGACCAAAAATCAGGTAAGTCTTACCTGCCTGGTAAAGGGTTTCTACCCCAGTGATATTGCCGTAGAGTGGGAATCTAATGGCCAGCCTGAAAATAACTACAAGACTACCCCACCAGTGCTTGACTCGGATGGGAGCTTCTTCCTGTACTCCCGCCTGACGGTTGACAAATCACGATGGCAGGAGGGCAATGTCTTCAGCTGTTCGGTCATGCACGAGGCTTTGCATAATCATTATACGCAAAAGTCCCTTTCCTTGAGTCTGGGCTGATTAATTAA
SEQ ID NO:13(Fulranumab轻链氨基酸,横线处表示CDR序列)
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:14(Fulranumab重链氨基酸,横线处表示CDR序列)
EVQLVESGGGLVQPGGSLRLSCAASGFTLRSYSMNWVRQAPGKGLEWVSYISRSSHTIFYADSVKGRFTISRDNAKNSLYLQMDSLRDEDTAMYYCARVYSSGWHVSDYFDYWGQGILVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:15(轻链中β-NGF的核苷酸序列)
TCTTCCAGCCATCCGATCTTCCATCGGGGAGAGTTCAGTGTCTGTGATAGCGTCTCAGTATGGGTGGGAGATAAGACAACAGCAACAGACATCAAAGGGAAAGAAGTCATGGTGCTAGGAGAGGTGAATATCAACAACTCTGTTTTCAAGCAGTATTTTTTTGAAACCAAGTGCAGAGATCCCAATCCTGTGGATTCGGGCTGCCGAGGTATCGACTCCAAGCACTGGAATAGCTACTGCACGACGACTCACACTTTTGTGAAAGCACTCACCATGGATGGAAAGCAAGCTGCCTGGAGGTTCATTCGGATTGACACCGCCTGTGTCTGCGTGCTGAGTCGGAAAGCTGTTCGATGA
SEQ ID NO:16(重链中β-NGF+Fc核苷酸序列)
TCTAGTTCTCATCCCATCTTCCACCGGGGGGAATTTAGTGTGTGTGACTCCGTGTCAGTGTGGGTGGGAGACAAGACCACCGCAACTGACATCAAGGGCAAGGAGGTGATGGTGCTGGGAGAGGTGAACATCAATAATTCAGTTTTCAAACAGTACTTCTTTGAAACAAAGTGTAGAGATCCGAACCCTGTTGATAGCGGCTGCAGGGGCATTGACAGCAAGCACTGGAACAGCTACTGCACCACGACACACACATTTGTGAAAGCGCTCACCATGGATGGGAAGCAGGCAGCCTGGCGCTTCATCCGGATTGATACAGCCTGCGTCTGTGTCCTAAGTAGAAAAGCTGTTCGAGGTGGCGGGGGCAGTGGCGGCGGCGGATCTGGAGGGGGTGGTTCCGGCGGGGGCGGCTCTGCCGAGAGCAAATATGGTCCTCCCTGCCCGCCGTGCCCAGCACCAGAAGCAGCTGGAGGTCCATCGGTGTTTCTCTTCCCACCCAAACCTAAAGATACACTGATGATCTCCCGGACACCAGAGGTCACTTGTGTGGTCGTGGATGTCTCCCAGGAAGACCCCGAGGTACAGTTCAACTGGTACGTGGACGGAGTGGAAGTGCACAACGCCAAAACCAAGCCTAGAGAAGAGCAGTTTAATAGCACGTATCGTGTAGTGTCCGTATTAACAGTCCTCCATCAAGACTGGCTCAACGGGAAAGAATATAAATGCAAGGTGAGCAACAAAGGACTCCCTTCTTCCATTGAGAAGACAATATCTAAGGCCAAGGGTCAGCCAAGAGAACCCCAAGTCTATACTCTGCCCCCGTCACAAGAAGAGATGACCAAAAATCAGGTAAGTCTTACCTGCCTGGTAAAGGGTTTCTACCCCAGTGATATTGCCGTAGAGTGGGAATCTAATGGCCAGCCTGAAAATAACTACAAGACTACCCCACCAGTGCTTGACTCGGATGGGAGCTTCTTCCTGTACTCCCGCCTGACGGTTGACAAATCACGATGGCAGGAGGGCAATGTCTTCAGCTGTTCGGTCATGCACGAGGCTTTGCATAATCATTATACGCAAAAGTCCCTTTCCTTGAGTCTGGGCTGA
序列表
<110> 三生国健药业(上海)股份有限公司
<120> 一种β-NGF融合蛋白、其制备方法及用途
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg
115
<210> 2
<211> 229
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 3
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 4
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
145 150 155 160
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
165 170 175
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
180 185 190
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
195 200 205
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
210 215 220
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
225 230 235 240
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
245 250 255
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
260 265 270
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
275 280 285
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
290 295 300
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
305 310 315 320
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
325 330 335
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
340 345 350
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
355 360 365
<210> 5
<211> 103
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln
1 5 10 15
Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg
20 25 30
Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln
35 40 45
Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu
50 55 60
Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala
65 70 75 80
Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn
85 90 95
Arg Thr His Arg Ser Lys Arg
100
<210> 6
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys
20
<210> 7
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 8
<211> 732
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggacacta gagcccccac ccagctgctg ggcctcttgc ttctctggct gccgggggcg 60
cgctgtgagc cccacagtga gagcaatgta cctgccgggc acaccattcc acaggcccac 120
tggactaaac tgcagcactc cctggataca gccttgcggc gcgcccgctc cgctcctgct 180
gctgcaatag cagcgcgggt ggccggccag accaggaaca taactgtgga ccctcgttta 240
tttaagaagc gcagactcag atctccaagg gttcttttct caacccaacc accaagagaa 300
gcagctgaca cacaggacct ggactttgag gtgggcggtg ccgcgccctt caacaggaca 360
cataggagca aacgctcttc cagccatccg atcttccatc ggggagagtt cagtgtctgt 420
gatagcgtct cagtatgggt gggagataag acaacagcaa cagacatcaa agggaaagaa 480
gtcatggtgc taggagaggt gaatatcaac aactctgttt tcaagcagta tttttttgaa 540
accaagtgca gagatcccaa tcctgtggat tcgggctgcc gaggtatcga ctccaagcac 600
tggaatagct actgcacgac gactcacact tttgtgaaag cactcaccat ggatggaaag 660
caagctgcct ggaggttcat tcggattgac accgcctgtg tctgcgtgct gagtcggaaa 720
gctgttcgat ga 732
<210> 9
<211> 1470
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggagactg ggctgaggtg gctgttacta gttgctgtcc tgaagggggt ccagtgtgag 60
ccccacagcg aaagcaatgt gccggcagga cacaccatac ctcaggccca ctggacaaag 120
ctgcagcatt ctttggacac tgctcttcgc agagcccggt ctgcacctgc cgctgccatc 180
gcagccaggg tggccggaca gactagaaac attacggttg acccccggct cttcaagaag 240
cgtcgccttc gcagcccccg ggtgctgttt tcaactcagc cccccaggga ggcggctgat 300
acccaagatc tggactttga ggttggagga gctgcgcctt ttaacaggac ccacaggtcc 360
aagcgatcta gttctcatcc catcttccac cggggggaat ttagtgtgtg tgactccgtg 420
tcagtgtggg tgggagacaa gaccaccgca actgacatca agggcaagga ggtgatggtg 480
ctgggagagg tgaacatcaa taattcagtt ttcaaacagt acttctttga aacaaagtgt 540
agagatccga accctgttga tagcggctgc aggggcattg acagcaagca ctggaacagc 600
tactgcacca cgacacacac atttgtgaaa gcgctcacca tggatgggaa gcaggcagcc 660
tggcgcttca tccggattga tacagcctgc gtctgtgtcc taagtagaaa agctgttcga 720
ggtggcgggg gcagtggcgg cggcggatct ggagggggtg gttccggcgg gggcggctct 780
gccgagagca aatatggtcc tccctgcccg ccgtgcccag caccagaagc agctggaggt 840
ccatcggtgt ttctcttccc acccaaacct aaagatacac tgatgatctc ccggacacca 900
gaggtcactt gtgtggtcgt ggatgtctcc caggaagacc ccgaggtaca gttcaactgg 960
tacgtggacg gagtggaagt gcacaacgcc aaaaccaagc ctagagaaga gcagtttaat 1020
agcacgtatc gtgtagtgtc cgtattaaca gtcctccatc aagactggct caacgggaaa 1080
gaatataaat gcaaggtgag caacaaagga ctcccttctt ccattgagaa gacaatatct 1140
aaggccaagg gtcagccaag agaaccccaa gtctatactc tgcccccgtc acaagaagag 1200
atgaccaaaa atcaggtaag tcttacctgc ctggtaaagg gtttctaccc cagtgatatt 1260
gccgtagagt gggaatctaa tggccagcct gaaaataact acaagactac cccaccagtg 1320
cttgactcgg atgggagctt cttcctgtac tcccgcctga cggttgacaa atcacgatgg 1380
caggagggca atgtcttcag ctgttcggtc atgcacgagg ctttgcataa tcattatacg 1440
caaaagtccc tttccttgag tctgggctga 1470
<210> 10
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gccacc 6
<210> 11
<211> 750
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aagcttgcca ccatggacac tagagccccc acccagctgc tgggcctctt gcttctctgg 60
ctgccggggg cgcgctgtga gccccacagt gagagcaatg tacctgccgg gcacaccatt 120
ccacaggccc actggactaa actgcagcac tccctggata cagccttgcg gcgcgcccgc 180
tccgctcctg ctgctgcaat agcagcgcgg gtggccggcc agaccaggaa cataactgtg 240
gaccctcgtt tatttaagaa gcgcagactc agatctccaa gggttctttt ctcaacccaa 300
ccaccaagag aagcagctga cacacaggac ctggactttg aggtgggcgg tgccgcgccc 360
ttcaacagga cacataggag caaacgctct tccagccatc cgatcttcca tcggggagag 420
ttcagtgtct gtgatagcgt ctcagtatgg gtgggagata agacaacagc aacagacatc 480
aaagggaaag aagtcatggt gctaggagag gtgaatatca acaactctgt tttcaagcag 540
tatttttttg aaaccaagtg cagagatccc aatcctgtgg attcgggctg ccgaggtatc 600
gactccaagc actggaatag ctactgcacg acgactcaca cttttgtgaa agcactcacc 660
atggatggaa agcaagctgc ctggaggttc attcggattg acaccgcctg tgtctgcgtg 720
ctgagtcgga aagctgttcg atgactcgag 750
<210> 12
<211> 1490
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ttcgaagcca ccatggagac tgggctgagg tggctgttac tagttgctgt cctgaagggg 60
gtccagtgtg agccccacag cgaaagcaat gtgccggcag gacacaccat acctcaggcc 120
cactggacaa agctgcagca ttctttggac actgctcttc gcagagcccg gtctgcacct 180
gccgctgcca tcgcagccag ggtggccgga cagactagaa acattacggt tgacccccgg 240
ctcttcaaga agcgtcgcct tcgcagcccc cgggtgctgt tttcaactca gccccccagg 300
gaggcggctg atacccaaga tctggacttt gaggttggag gagctgcgcc ttttaacagg 360
acccacaggt ccaagcgatc tagttctcat cccatcttcc accgggggga atttagtgtg 420
tgtgactccg tgtcagtgtg ggtgggagac aagaccaccg caactgacat caagggcaag 480
gaggtgatgg tgctgggaga ggtgaacatc aataattcag ttttcaaaca gtacttcttt 540
gaaacaaagt gtagagatcc gaaccctgtt gatagcggct gcaggggcat tgacagcaag 600
cactggaaca gctactgcac cacgacacac acatttgtga aagcgctcac catggatggg 660
aagcaggcag cctggcgctt catccggatt gatacagcct gcgtctgtgt cctaagtaga 720
aaagctgttc gaggtggcgg gggcagtggc ggcggcggat ctggaggggg tggttccggc 780
gggggcggct ctgccgagag caaatatggt cctccctgcc cgccgtgccc agcaccagaa 840
gcagctggag gtccatcggt gtttctcttc ccacccaaac ctaaagatac actgatgatc 900
tcccggacac cagaggtcac ttgtgtggtc gtggatgtct cccaggaaga ccccgaggta 960
cagttcaact ggtacgtgga cggagtggaa gtgcacaacg ccaaaaccaa gcctagagaa 1020
gagcagttta atagcacgta tcgtgtagtg tccgtattaa cagtcctcca tcaagactgg 1080
ctcaacggga aagaatataa atgcaaggtg agcaacaaag gactcccttc ttccattgag 1140
aagacaatat ctaaggccaa gggtcagcca agagaacccc aagtctatac tctgcccccg 1200
tcacaagaag agatgaccaa aaatcaggta agtcttacct gcctggtaaa gggtttctac 1260
cccagtgata ttgccgtaga gtgggaatct aatggccagc ctgaaaataa ctacaagact 1320
accccaccag tgcttgactc ggatgggagc ttcttcctgt actcccgcct gacggttgac 1380
aaatcacgat ggcaggaggg caatgtcttc agctgttcgg tcatgcacga ggctttgcat 1440
aatcattata cgcaaaagtc cctttccttg agtctgggct gattaattaa 1490
<210> 13
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 14
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
195 200 205
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys
210 215 220
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
290 295 300
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 15
<211> 357
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tcttccagcc atccgatctt ccatcgggga gagttcagtg tctgtgatag cgtctcagta 60
tgggtgggag ataagacaac agcaacagac atcaaaggga aagaagtcat ggtgctagga 120
gaggtgaata tcaacaactc tgttttcaag cagtattttt ttgaaaccaa gtgcagagat 180
cccaatcctg tggattcggg ctgccgaggt atcgactcca agcactggaa tagctactgc 240
acgacgactc acacttttgt gaaagcactc accatggatg gaaagcaagc tgcctggagg 300
ttcattcgga ttgacaccgc ctgtgtctgc gtgctgagtc ggaaagctgt tcgatga 357
<210> 16
<211> 1104
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tctagttctc atcccatctt ccaccggggg gaatttagtg tgtgtgactc cgtgtcagtg 60
tgggtgggag acaagaccac cgcaactgac atcaagggca aggaggtgat ggtgctggga 120
gaggtgaaca tcaataattc agttttcaaa cagtacttct ttgaaacaaa gtgtagagat 180
ccgaaccctg ttgatagcgg ctgcaggggc attgacagca agcactggaa cagctactgc 240
accacgacac acacatttgt gaaagcgctc accatggatg ggaagcaggc agcctggcgc 300
ttcatccgga ttgatacagc ctgcgtctgt gtcctaagta gaaaagctgt tcgaggtggc 360
gggggcagtg gcggcggcgg atctggaggg ggtggttccg gcgggggcgg ctctgccgag 420
agcaaatatg gtcctccctg cccgccgtgc ccagcaccag aagcagctgg aggtccatcg 480
gtgtttctct tcccacccaa acctaaagat acactgatga tctcccggac accagaggtc 540
acttgtgtgg tcgtggatgt ctcccaggaa gaccccgagg tacagttcaa ctggtacgtg 600
gacggagtgg aagtgcacaa cgccaaaacc aagcctagag aagagcagtt taatagcacg 660
tatcgtgtag tgtccgtatt aacagtcctc catcaagact ggctcaacgg gaaagaatat 720
aaatgcaagg tgagcaacaa aggactccct tcttccattg agaagacaat atctaaggcc 780
aagggtcagc caagagaacc ccaagtctat actctgcccc cgtcacaaga agagatgacc 840
aaaaatcagg taagtcttac ctgcctggta aagggtttct accccagtga tattgccgta 900
gagtgggaat ctaatggcca gcctgaaaat aactacaaga ctaccccacc agtgcttgac 960
tcggatggga gcttcttcct gtactcccgc ctgacggttg acaaatcacg atggcaggag 1020
ggcaatgtct tcagctgttc ggtcatgcac gaggctttgc ataatcatta tacgcaaaag 1080
tccctttcct tgagtctggg ctga 1104
Claims (19)
1.一种β-NGF融合蛋白,其特征在于,包含:
(a)轻链,所述轻链包含β-NGF;和
(b)重链,所述重链包含β-NGF和Fc片段。
2.如权利要求1所述的β-NGF融合蛋白,其特征在于,所述β-NGF为人β-NGF;优选地,所述β-NGF包含如SEQ ID NO:1所示氨基酸序列。
3.如权利要求1所述的β-NGF融合蛋白,其特征在于,所述β-NGF和所述Fc片段通过连接子连接。
4.如权利要求3所述的β-NGF融合蛋白,其特征在于,所述重链从N端到C端依次为β-NGF、连接子和Fc片段。
5.如权利要求3或4所述的β-NGF融合蛋白,其特征在于,所述连接子包含通式(GGGGS)n,n为整数,n=1~10;较佳地n=4~10;优选地n=4,氨基酸序列如SEQ ID NO:3所示。
6.如权利要求1所述的β-NGF融合蛋白,其特征在于,所述Fc片段来源于IgG。
7.如权利要求6所述的β-NGF融合蛋白,其特征在于,所述IgG来源于哺乳动物;较佳地来源于鼠、食蟹猴或人;优选来源于人。
8.如权利要求6所述的β-NGF融合蛋白,其特征在于,所述Fc片段选自IgG1、IgG2、IgG3或IgG4;较佳地选自IgG4。
9.如权利要求6所述的β-NGF融合蛋白,其特征在于,所述Fc片段包含人IgG铰链区、CH2和CH3区;较佳地,包含人IgG4铰链区、CH2和CH3区;优选地,包含如SEQ ID NO:2所示氨基酸序列。
10.如权利要求1所述的β-NGF融合蛋白,其特征在于,所述Fc片段包含Fc变体,所述Fc变体包含一个或多个氨基酸的取代、缺失和/或插入。
11.如权利要求1所述的β-NGF融合蛋白,其特征在于,所述轻链包含如SEQ ID NO:1所示的氨基酸序列;和/或所述重链包含如SEQ ID NO:4所示的氨基酸序列。
12.一种分离的核酸分子,其特征在于,所述核酸分子包含编码如权利要求1-11中任一项所述的β-NGF融合蛋白的核苷酸序列。
13.如权利要求12所述的核酸分子,其特征在于,(1)所述核酸分子包含如SEQ ID NO:8所示轻链核苷酸序列和如SEQ ID NO:9所示重链核苷酸序列;(2)所述核酸分子包含如SEQID NO:11所示轻链核苷酸序列和如SEQ ID NO:12所示重链核苷酸序列;或(3)所述核酸分子包含如SEQ ID NO:15所示轻链核苷酸序列和如SEQ ID NO:16所示重链核苷酸序列。
14.一种表达载体,其特征在于,所述的表达载体包含如权利要求12或13所述的核酸分子。
15.一种宿主细胞,其特征在于,所述宿主细胞包含如权利要求14所述的表达载体。
16.如权利要求1-11中任一项所述的β-NGF融合蛋白的制备方法,其特征在于,所述方法包括:
(a)在表达条件下,培养如权利要求15所述的宿主细胞,从而表达β-NGF融合蛋白;
(b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
17.一种药物组合物,其特征在于,所述药物组合物包含如权利要求1-11中任一项所述的β-NGF融合蛋白和药学上可接受的载体。
18.如权利要求1-11中任一项所述的β-NGF融合蛋白或权利要求17所述的药物组合物,其与另外的治疗剂分别、依次或同时使用。
19.如权利要求1-11中任一项所述的β-NGF融合蛋白或权利要求18所述的药物组合物用于制备治疗中度至重度神经营养性角膜炎或者外周神经损伤药物的用途。
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