CN105085652B - 血小板衍生生长因子b突变体、其制备方法及用途 - Google Patents
血小板衍生生长因子b突变体、其制备方法及用途 Download PDFInfo
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Abstract
本发明涉及血小板衍生生长因子B衍生物,具体地,本发明涉及血小板衍生生长因子B突变体,编码该突变体的核酸分子,含有该核酸分子的载体和宿主细胞。本发明还涉及所述突变体的制备方法和纯化方法,以及所述突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
Description
技术领域
本发明涉及血小板衍生生长因子B衍生物,具体地,本发明涉及血小板衍生生长因子B突变体,编码该突变体的核酸分子,含有该核酸分子的载体和宿主细胞。本发明还涉及所述突变体的制备方法和纯化方法,以及所述突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
背景技术
血小板衍生生长因子(Platelet-derived growth factor,PDGF)是一种能多种细胞产生的、能够刺激间质来源细胞增殖的多肽,上世纪70年代首先由Ross等从血小板中发现,因而得名(1)。到目前为止,共发现4种PDGF单体PDGF-A、PDGF-B、PDGF-C和PDGF-D。这些单体彼此通过链内及链间二硫键,形成5种同源或异源二聚体:PDGF-AA、PDGF-BB、PDGF-AB、PDGF-CC和PDGF-DD(2,3)。普遍认为PDGF基因和蛋白属于一类在结构与功能上相关的生长因子家族,这个家族还包括血管内皮生长因子(VEGFs)以及胎盘生长因子(PIGF)(4)。PDGF通过激活其受体PDGF-R发挥生理功能。PDGF-R包括PDGFR-α和PDGFR-β两种,属于酪氨酸激酶受体。配体与受体结合引发受体单体的二聚化,促使胞内区的酪氨酸残基自体磷酸化而激活。两种受体可以激活多条信号通路关键分子,如Ras-MAPK、PI3K和PLC-γ(5),进而激活相关基因的转录,刺激细胞生长、抑制凋亡,促进分化,引起定向移动和迁移等,发挥多种多样的生物学功能。
PDGF-b基因定位于第22号染色体,含有7个外显子基因,编码241个氨基酸组成的前体蛋白,经蛋白酶水解加工形成的最终成熟产物是109个氨基酸组成的,分子量为12.3kD的多肽。在生物体内,PDGF-B蛋白的活性形式是由两条单体通过二硫键形成同源PDGF-BB或异源二聚体PDGF-AB(6)。每条PDGF-B蛋白单体含有8个高度保守的半胱氨酸残基,其中6个半胱氨酸两两形成链内二硫键(CysⅠ-Ⅵ,Ⅲ-Ⅶ,Ⅴ-Ⅷ),另外两个则与对应单体之间交叉形成链间二硫键(CysⅡ-Ⅳ)(7)共同形成PDGF蛋白家族特征性的生长因子结构域—半胱氨酸结。这些链内及链间二硫键构成了PDGF-BB二聚体蛋白复杂的空间结构(图1B)。
另外,PDGF蛋白在表达合成时还存在不同剪切形式,使得加工成熟后的PDGF蛋白呈现多种结构形式。从人血小板提取物中分离纯化的PDGF-BB,通过N端氨基酸序列分析表明存在至少三种不同的剪切形式,20%Ser1,45%Thr6及35%Thr33,这些切割的异质性,致使在纯化PDGF-BB时各种切割形式的蛋白比例不可控(8)。
发明内容
本发明的发明人经过大量研究,发现特定位点蛋白酶降解和/或糖基化修饰是造成多种PDGF-B存在的主要原因,进而通过位点突变获得了PDGF-B突变体,其蛋白均一性大大提高,并且仍保留PDGF-B蛋白的活性。
本发明第一方面涉及血小板衍生生长因子B突变体,其在野生型血小板衍生生长因子B的第101位和第109位氨基酸位点处具有突变(此处对氨基酸位点位置的描述均以含109个氨基酸残基的成熟PDGF-B为基础,以下同),并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,其在第6位氨基酸位点处具有突变,并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,其在第32位和/或第33位氨基酸位点处具有突变,并且具有血小板衍生生长因子B的活性。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,与野生型血小板衍生生长因子B相比,其N端缺失5个氨基酸,并且具有血小板衍生生长因子B的活性。
在本发明的一个实施方案中,所述突变体在第6位、第101位和第109位氨基酸位点处突变为丙氨酸。
在本发明的另一个实施方案中,所述突变体在第101位和第109位氨基酸位点处突变为丙氨酸。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,其在第32位和/或第33位氨基酸位点处突变为脯氨酸、缬氨酸或异亮氨酸。
在本发明的一个实施方案中,其在第32位氨基酸位点处突变为脯氨酸、缬氨酸或异亮氨酸,优选为脯氨酸。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,其中所述的血小板衍生生长因子B为哺乳动物来衍生的血小板衍生生长因子B,所述哺乳动物例如为人、小鼠。
在本发明的一个实施方案中,所述血小板衍生生长因子B突变体为,其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中,所述血小板衍生生长因子B突变体为,其N端缺失5个氨基酸,其第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中,所述血小板衍生生长因子B突变体为,其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为缬氨酸。
在本发明的一个实施方案中,所述血小板衍生生长因子B突变体为,其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为异亮氨酸。
本发明还包括上述各种技术方案的组合。
在本发明的具体实施方案中,所述突变体的氨基酸序列为SEQ IDNO:3或SEQ IDNO:5所示的序列。
根据本发明第一方面任一项的血小板衍生生长因子B突变体,其氨基酸序列经过取代、缺失或添加一个几个氨基酸,并且具有血小板衍生生长因子B的活性。
本发明第二方面涉及血小板衍生生长因子同源或异源二聚体,其由两个本发明第一方面任一项的血小板衍生生长因子B突变体通过链内及链间二硫键结合而成,或者由一个本发明第一方面任一项的血小板衍生生长因子B突变体与一个血小板衍生生长因子A通过链内或链间二硫键结合而成。
在本发明中,所述血小板衍生生长因子同源或异源二聚体的形成方式与野生型血小板衍生生长因子相同。
在本发明的实施方案中,两个本发明第一方面任一项的血小板衍生生长因子B突变体通过链内及链间二硫键结合,形成PDGF-BB突变体。
本发明第三方面涉及核酸分子,其编码本发明第一方面任一项的血小板衍生生长因子B突变体。
根据本发明第三方面任一项的核酸分子,其核苷酸序列选自SEQ IDNO:4、SEQ IDNO:6~SEQ ID NO:9的序列。
本发明第四方面涉及载体,其含有本发明第三方面任一项的核酸分子。
在本发明中,本领域技术人员可以根据用于表达的宿主细胞的不同来选择表达载体,例如可以选择适合酵母细胞或哺乳动物细胞表达的载体。
在本发明的实施方案中,所述载体为pMEX9K。
本发明第五方面涉及宿主细胞,其含有本发明第四方面任一项的载体。
根据本发明第五方面任一项的宿主细胞,其为真核细胞,例如为酵母细胞、哺乳动物细胞或昆虫细胞。
根据本发明第五方面任一项的宿主细胞,所述酵母细胞例如为毕赤酵母细胞(Pichia pastoris,也叫巴斯德毕赤酵母细胞)、酿酒酵母(Saccharomyces cerevisiae)、克鲁维酵母(Kluyveromyces lactis)、汉逊酵母(Hansenula)、念珠酵母(Candida)或球拟酵母(Torulopsis)。
在本发明的实施方案中,所述毕赤酵母细胞为GS115细胞。
根据本发明第五方面任一项的宿主细胞,所述哺乳细胞例如为CHO细胞、BHK细胞、NS0细胞、SP2/0细胞、HEK-293细胞、COS细胞等。
本发明还涉及本发明第一方面任一项的血小板衍生生长因子B突变体的制备方法,其包括取本发明第五方面任一项的宿主细胞进行培养、表达(例如诱导表达)以及任选的纯化的步骤。
根据本发明任一项的制备方法,其包括以下步骤:
1)取本发明第五方面任一项的宿主细胞接种至培养基中,经过逐级放大培养;
2)收集宿主细胞,将重组细胞重悬于培养基中,添加甲醇开始诱导表达;
3)诱导表达结束后收集培养上清,经纯化获得血小板衍生生长因子B蛋白。
根据本发明任一项的制备方法,其特征在于以下几项中的一项或数项:
(1)步骤1)中的宿主细胞为单克隆细胞株;
(2)步骤1)中所述的逐级放大培养是指两级放大培养,培养温度为28-30℃,每级均培养至OD600为1-12、例如2-6;
(3)步骤2)中诱导表达的温度约为28℃;
(4)步骤2)中甲醇的终浓度为0.3-1.0%(v/v),例如0.4-0.8%(v/v),例如0.5%(v/v);
(5)步骤2)中诱导表达的时间为48-96h,例如为72h;
(6)步骤3)中的纯化步骤依次包括疏水层析、离子交换层析、凝胶层析。
本发明第六方面涉及血小板衍生生长因子B或其突变体的纯化方法,其包括取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液依次进行疏水层析、离子交换层析和凝胶层析的步骤。
根据本发明第六方面任一项的纯化方法,其中所述血小板衍生生长因子B突变体为本发明第一方面任一项的血小板衍生生长因子B突变体。
在本发明的实施方案中,用于疏水层析的层析柱介质为Phenyl Sepharose6FastFlow。
在本发明的实施方案中,用于离子交换层析的层析介质为Source30S。
在本发明的实施方案中,用于凝胶层析的层析介质为Hiload Superdex75prepgrad。
根据本发明第六方面任一项的纯化方法,其中,
所述的疏水层析包括以下步骤:
(1)取含有血小板衍生生长因子B或其突变体的培养上清或细胞裂解液用调节缓冲液调节电导,所述调节缓冲液加入后的终体系为10-50mM磷酸缓冲液,0.8-1M(NH4)2SO4,pH6.8-7.5;
(2)用平衡缓冲液平衡柱子,所述平衡缓冲液的配方为10-50mM磷酸缓冲液,0.8-1M(NH4)2SO4,pH6.8-7.5;
(3)样品上柱后,用平衡缓冲液洗柱;
(4)用洗脱缓冲液洗脱,收集目的蛋白,所述洗脱缓冲液的配方为,10-50mM磷酸缓冲液,30%-50%乙二醇,pH6.8-7.5;
所述离子交换层析包括以下步骤:
(1)用平衡缓冲液稀释疏水层析的洗脱峰至电导值为6mS/cm以下,所述平衡缓冲液的配方为,10-50mM磷酸缓冲液,pH6.8-7.5;
(2)用平衡缓冲液平衡柱子;
(3)样品上柱后,用平衡缓冲液清洗柱子;
(4)用洗脱缓冲液梯度洗脱,收集目的蛋白,所述洗脱缓冲液的配方为10-50mM磷酸缓冲液,0.8-1.2M NaCl,pH6.8-7.5;
所述凝胶层析包括以下步骤:
(1)用磷酸盐缓冲液平衡柱子,所述磷酸盐缓冲液的配方为,10-50mM磷酸缓冲液,0.1-0.5M NaCl,pH6.8-7.5;
(2)将离子交换层析的洗脱峰上样,每次上样体积不超过柱体积的0.3-4%(例如3%);
(3)继续用步骤(1)中的磷酸盐缓冲液清洗柱子,收集目的蛋白,即得到纯化的血小板衍生生长因子B或其突变体。在本发明中,所述磷酸缓冲液的配方为本领域所公知。在本发明的实施方案中,所述磷酸缓冲液为20mMPB溶液,其含有0.0144mol/L的Na2HPO4和0.0056mol/L的NaH2PO4,pH6.8-7.5。
在本发明中,所述磷酸盐缓冲液的配方为本领域所公知。在本发明的实施方案中,所述磷酸盐缓冲液为PBS溶液,其配方为10-50mM PB溶液,0.15M NaCl,pH6.8-7.5。
在本发明的实施方案中,各步层析的缓冲液的pH值为7.2。
在本发明的实施方案中,各步层析的磷酸缓冲液的浓度为20mM。
在本发明的具体实施方案中,疏水层析方法为:(1)酵母表达上清用1/2体积的调节缓冲液(60mM PB,3M(NH4)2SO4,pH7.2)调节电导;(2)用平衡缓冲液(20mM PB,1M(NH4)2SO4,pH7.2)平衡柱子;(3)样品上柱后,用平衡缓冲液清洗柱子至基线平直;(4)用洗脱缓冲液(20mM PB,50%乙二醇,pH7.2)洗脱,收集目的蛋白。
在本发明的具体实施方案中,离子交换层析方法为:(1)用平衡缓冲液(20mM PB,pH7.2)稀释Phenyl HS洗脱峰至电导值为6mS/cm以下;(2)用平衡缓冲液平衡柱子;(3)样品上柱后,用平衡缓冲液清洗柱子至基线平直;(4)用洗脱缓冲液(20mM PB,1M NaCl,pH7.2)梯度洗脱,收集目的蛋白。
在本发明的具体实施方案中,凝胶层析方法为:(1)用PBS缓冲液(20mMPB,0.15MNaCl,pH7.2)平衡柱子;(2)将Source30S洗脱峰用loop上样,每次上样体积不超过柱体积的3%;(3)继续用PBS缓冲液清洗柱子,收集目的蛋白。
本发明还涉及本发明第一方面任一项的血小板衍生生长因子B突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
本发明还涉及抗体,其能够与本发明第一方面任一项的血小板衍生生长因子B突变体特异性结合。
本发明还涉及一种使血小板衍生生长因子表达更均一化的方法,其包括对野生型血小板衍生生长因子的氨基酸序列进行改造的步骤,所述改造包括以下a)-c)中的一项或几项:
a)突变第101位和第109位氨基酸;
b)突变第6位氨基酸;
c)突变第32位和/或第33位氨基酸;
d)N端缺失5个氨基酸。
根据本发明任一项的方法,其特征在于以下i)-iii)中的一项或几项:
i)将第101位和第109位氨基酸突变为丙氨酸;
ii)将第6位氨基酸突变为丙氨酸;
iii)将第32位和/或第33位氨基酸突变为脯氨酸、缬氨酸或异亮氨酸。
根据本发明任一项的方法,利用真核表达系统进行表达,如酵母细胞表达系统、哺乳动物细胞表达系统。
在本发明的一个实施方案中,所述改造是指其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中,所述改造是指其N端缺失5个氨基酸,其第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为脯氨酸。
在本发明的一个实施方案中,所述改造是指其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为缬氨酸。
在本发明的一个实施方案中,所述改造是指其N端缺失5个氨基酸,其第6位、第101位和第109位氨基酸突变为丙氨酸,其第32位氨基酸突变为异亮氨酸。
在本发明的具体实施方案中,改造后的血小板衍生生长因子的氨基酸序列为SEQID NO:3或SEQ ID NO:5所示的序列。
本发明还包括上述各种技术方案的组合。
在本发明中,所述载体例如为克隆载体或表达载体。所述载体含有本发明所述的核酸分子,所述载体可以通过例如将上述核酸分子插入克隆载体或表达载体而得到,或者可以通过人工合成得到。
所述表达载体例如为原核表达载体,真核表达载体,噬菌体载体或病毒载体。其中所述原核表达载体例如为pET载体、pGEX载体,所述真核表达载体例如为pcDNA3.1、pEGFP-C1、pPIC9K、pMEX9K、pPICZ、pPICZa、pFastBac、pPIC6aA、pPIC3.5K、pGAPZaA、pAO815,所述噬菌体载体例如为λ噬菌体载体λgt、λgt-λB,所述病毒载体例如为逆转录病毒、慢病毒、腺病毒或腺相关病毒载体。在本发明的实施方案中,所述载体为pMEX9K。
在本发明中,所述宿主细胞可以为原核细胞(例如大肠杆菌细胞)或真核细胞。所述真核细胞例如为酵母细胞、哺乳动物细胞或昆虫细胞。所述宿主细胞可以通过向原核细胞或真核细胞中引入/转染上述的核酸分子的核苷酸序列或载体而得到。
在本发明中,所述酵母细胞株为本领域所公知,其包括但不限于X33,KM71,KM71H,GS115,SMD1168,SMD1163,SMD1168H,SMD1163H。
在本发明中,可以利用本领域所知的任何种类的转染方法获得转染有特定核酸或载体的宿主细胞,例如,可通过电穿孔或显微注射将核酸引入细胞中;或者,可使用脂转染试剂如FuGENE6、X-tremeGENE和LipofectAmine;或者,可通过基于逆转录病毒、慢病毒、腺病毒和腺相关病毒的适当病毒病毒载体将核酸引入细胞中。
在本发明中,所述表达均一化是指在电泳上样量不低于10μg时,经还原型SDS-PAGE电泳分析,目的蛋白经考马斯亮蓝染色为单一条带,Image J或Bandscan等软件分析纯度>95%。本领域技术人员通过摸索,针对不同的蛋白,采取各种技术手段,可以使蛋白的表达趋向于更均一化。
在本发明中,所述野生型血小板衍生生长因子B是指长度为109个氨基酸的未经过突变的血小板衍生生长因子B;并且对各位点(例如突变位点、糖基化位点、酶切位点)的位置描述也均是以野生型血小板衍生生长因子B为基准。
在本发明中,所述第6位、第101位、第109位氨基酸均为苏氨酸(Thr);所述第32位氨基酸、第33位氨基酸分别为精氨酸(Arg);野生型血小板衍生生长因子B的N端前5个氨基酸依次为丝氨酸(Ser)、亮氨酸(Leu)、甘氨酸(Gly)、丝氨酸(Ser)、亮氨酸(Leu)。
本发明通过突变获得了PDGF-B突变体,其蛋白均一性大大提高,并且仍保留了PDGF-B蛋白的活性。
附图说明
图1.PDGF-B蛋白结构示意图。A,PDGF-B前体蛋白通过蛋白酶水解掉N端信号肽、前肽及C端前肽序列,成为成熟蛋白。▲表示蛋白酶水解位点;B,两个PDGF-B单体通过链间二硫键形成PDGF-BB同源二聚体,每个PDGF-B单体含有8个Cys,分别形成3对链内二硫键(C16-C60、C49-C97、C53-C99)及2对链间二硫键(C43-C52、C52-C43)。SP:信号肽(signalpeptide);PRO:生长因子结构域前序列(the pro-sequence preceding the growthfactor domain)。
图2.SDS-PAGE电泳分析毕赤酵母分泌表达的PDGF-BB。对3个不同批次发酵纯化的重组PDGF-BBThr6进行非还原条件(左)及还原条件(右)SDS-PAGE分析。非还原条件下蛋白为单一条带;经DTT处理后PDGF-B单体表现为分子量不均一的多种形式。
图3蛋白酶解及糖基化修饰共同作用致使PDGF-BThr6多种单体的形成。(A)对还原条件下SDS-PAGE分离PDGF-BThr6蛋白进行N-末端氨基酸序列分析。第1、2、3条带N端前5个氨基酸残基为均为TIATP,第4、5带N端前5个氨基酸残基为TNANF,表明在Arg32-Thr33发生蛋白酶切割。(B)对还原条件下PDGF-BThr6蛋白进行WB(中)及糖蛋白染色(右)分析。WB检测条带对应考马斯亮蓝染色(左)第3、5条带;糖蛋白染色条带对应考染第1、2、4条带。(C)利用PNGase F对还原条件下PDGF-BThr6蛋白进行处理并进行糖蛋白染色。蛋白经PNGase F处理前后带型、分子量无变化(左),糖蛋白染色结果无差别;IFN-ω为糖苷酶酶切及糖蛋白染色阳性对照。
图4.PDGF-BThr6蛋白序列O-连接糖基化位点预测结果。Thr6、Thr101、Thr109为可能的O-连接糖基化修饰位点。
图5HPLC检测纯化后蛋白PDGF-M2纯度结果。
图6毕赤酵母表达PDGF-B发生翻译后修饰位点分析。(A)PDGF-M1及PDGF-M2突变体位点突变示意图。(B)WB检测显示PDGF-M1及PDGF-M2单体为单一条带,对照PDGF-BThr6为两条。(C)SDS-PAGE检测PDGF-M1及PDGF-M2单体,经考马斯亮蓝染色结果显示PDGF-M2为单一蛋白条带,PDGF-M1为两条蛋白条带(如图中箭头所示)。(D)糖蛋白染色几乎检测不到PDGF-M1及PDGF-M2蛋白单体。
图7PDGF-M2的生物学活性高于PDGF-BBThr6,两者有统计学差异。实验重复3次,EC50表示为平均值±标准差,P值=0.039。
图8Arg32为突变对表达量的影响。(A)试管表达密码子优化株PDGF-IM-P、PDGF-IM-V、PDGF-IM-I各7个克隆的SDS-PAGE结果。PDGF-IM-P的蛋白表达量明显高于其他两株。(B)通过G418抗性对密码子优化株PDGF-IM-P、PDGF-IM-V、PDGF-IM-I分别进行多插入子拷贝筛选,分别选取G418浓度为2.0mg/ml及4.0mg/ml条件下筛选菌株6个克隆进行试管表达,SDS-PAGE结果显示,PDGF-IM-P高拷贝筛选株表达量明显高于其他两株。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
在前期研究中,我们成功利用毕赤酵母系统分泌表达了N端缺失5个氨基酸的rhPDGF-BBThr6,表达量可达100mg/L(参见专利:ZL200410068993.2)。选择PDGF-BThr6作为研究对象,是为了保证表达蛋白的均一性而生物活性又不受损。然而进一步研究发现,毕赤酵母所表达的rhPDGF-BThr6单体仍表现为分子量不均一的多种形式,分子量在10-15kDa之间(图2)。
以下实施例均是在rhPDGF-BThr6的基础上进行的改造,所有对位点位置的描述均是以野生型PDGF-B(109个氨基酸)为基础。
其中野生型PDGF-B氨基酸序列的Genbank号为NM-002608.2。
rhPDGF-BBThr6氨基酸序列为:
TIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT(SEQ ID NO:1)。
rhPDGF-BBThr6核酸序列为:
5’-ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCGCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGACAGTGGCAGCTGCACGGCCTGTGACC-3’(SEQ ID NO:2)。
材料和方法
重组表达克隆的构建
编码各种PDGF突变体DNA序列均由上海生工合成。基因片段通过酶切位点XhoI和EcoRI克隆至表达载体pMEX9K(参见专利ZL02117906.9)中,并经测序确认。提取重组质粒,经SalI酶切线性化后,利用电转化方法转化至毕赤酵母(Pichia pastoris)表达菌株GS115感受态细胞中。经组氨酸缺陷的MD平板筛选酵母转化子,并经PCR方法鉴定阳性重组酵母菌株。
重组蛋白的诱导表达
将重组酵母菌株单克隆接种至含有25mL BMGY培养基(BMGY培养基配方:称量酵母浸出粉10g,胰蛋白胨20g,溶于700ml水中,121℃高压灭菌20min;冷却至室温,加入100ml1M磷酸钾缓冲液、100ml10×YNB、100ml10×GY,4℃存放。其中:10×YNB(13.4%酵母氮源碱)、10×GY(10%甘油)、1M磷酸钾缓冲液:量取132ml1M K2HPO4,868ml1M KH2PO4,用磷酸或KOH调pH值至6.0±0.1,121℃高压灭菌30min,室温存放。YeastExtract(LP0021)为OXOID公司产品,Peptone(211677)为B&D公司产品)的摇瓶中,28-30℃,220-250rpm培养增菌至OD600=2-6(约16-18小时);将25ml酵母培养物接种至含有1L BMGY的摇瓶中,继续28-30℃,220-250rpm培养增菌至OD600=2-6;室温1500-3000g离心5分钟收集酵母,去除上清后用1L BMMY培养基重悬酵母开始诱导表达。诱导温度为28℃,摇床转速为220rpm;每24小时添加甲醇至终浓度为0.5%,共诱导72小时。诱导结束后室温7000rpm离心收集含重组蛋白的上清。
重组蛋白的纯化
毕赤酵母表达上清经离心、过滤后调整至合适的缓冲液中,依次经过疏水层析(Phenyl Sepharose6Fast Flow)、离子交换层析(Source30S)、凝胶层析(HiloadSuperdex75prep grad)获得纯度>95%的目的蛋白(图5)。层析介质均为GE AmershamBioscience公司产品。
疏水层析方法为:(1)酵母表达上清用1/2体积的调节缓冲液(60mMPB,3M(NH4)2SO4,pH7.2)调节电导;(2)按照说明书方法,用平衡缓冲液(20mM PB,1M(NH4)2SO4,pH7.2)平衡柱子;(3)样品上柱后,用平衡缓冲液清洗柱子至基线平直;(4)用洗脱缓冲液(20mM PB,50%乙二醇,pH7.2)洗脱,收集目的蛋白。
离子交换层析方法为:(1)用平衡缓冲液(20mM PB,pH7.2)稀释Phenyl HS洗脱峰至电导值为6mS/cm以下;(2)按照说明书方法,用平衡缓冲液平衡柱子;(3)样品上柱后,用平衡缓冲液清洗柱子至基线平直;(4)用洗脱缓冲液(20mM PB,1M NaCl,pH7.2)梯度洗脱,收集目的蛋白。
凝胶层析方法为:(1)用PBS缓冲液(20mM PB,0.15M NaCl,pH7.2)平衡柱子;(2)将Source30S洗脱峰用loop上样,每次上样体积不超过柱体积的3%;(3)继续用PBS缓冲液清洗柱子,收集目的蛋白。
重组蛋白SDS-PAGE检测
取30μl合适浓度的纯化后蛋白分别加入10μl4×SDS-PAGE上样缓冲液(不含及含20mM DTT),100℃变性5分钟,离心后取30μl上清经SDS-PAGE电泳分析(分离胶为15%)。电泳后凝胶用考马斯亮蓝R250染色。
重组蛋白Western Blot(WB)检测
样品制备同SDS-PAGE,取3μl样品进行SDS-PAGE,电泳结束后,300mA稳流电转1h,将蛋白转移至硝酸纤维素膜上,5%脱脂奶粉/TBST室温封闭1h。1:1000稀释一抗PDGF-B(F-3)(Santa Cruz Biotechnology,SC-365805),室温包被1h,TBST洗涤多次后,1:10000稀释HRP标记的二抗(Cell SignalingTechnology,#7076)室温孵育1h,TBST洗涤后,加入底物并用LAS400mini凝胶成像系统(GE)成像。
N-末端氨基酸序列测序
样品制备及SDS-PAGE过程同前,电泳结束后,用CAPS电印迹缓冲液,300mA稳流电转1h,将蛋白转移至PVDF膜上,0.1%考马斯亮蓝R250染色后立即用50%甲醇充分脱色至蛋白条带清洗可见,剪下待测蛋白条带委托军事医学科学院生物医学分析中心色谱实验室测定。
蛋白糖基化修饰检测
取5μl样品及阳性对照IFN-ω,加入3μl10×糖蛋白变性缓冲液(NEBPNGase F酶自带)及15μl水后100℃加热变性10min。冷却后加入3μl NP-40、3μl G7缓冲液(NEB PNGase F酶自带)及2μl肽N-糖苷酶F(PNGase F)(纽英伦生物技术有限公司(NEB)产品),37℃酶切3h。酶切结束后100℃加热样品使酶失活后进行SDS-PAGE电泳。电泳结束后,利用糖蛋白染色试剂盒(Themo Scientific,#24562)进行染色。首先加入100ml50%甲醇固定凝胶30min;3%乙酸清洗多次后将胶移入25ml Oxidizing Solution,轻摇15min;3%乙酸清洗多次后将胶移入25ml Glycoprotein Staining Reagent,轻摇15min后将胶移入25ml ReducingSolution,轻摇5min后用3%乙酸清洗并用去离子水漂洗。
PDGF-B生物学活性检测
BALB/C3T3细胞(购自北京协和细胞资源中心)用含10%FBS的DMEM完全培养基(Life Technology)于37℃、5%二氧化碳条件下培养。消化和收集细胞后,用完全培养液配成每1ml含5.0×104个细胞的细胞悬液,接种于96孔细胞培养板中,每孔100μl,于37℃、5%二氧化碳条件下继续培养;24h后换成维持培养基(含0.4%FBS的DMEM),于37℃、5%二氧化碳条件下继续培养;培养24h后弃去维持培养液,加入预先梯度稀释好的重组PDGF-BB溶液,每孔100μl;细胞在蛋白作用下继续培养64-72h后,采用WST-1法检测细胞增殖活性:每孔加入WST-1溶液(Roche,11644807001)10μl,于37℃、5%二氧化碳条件下继续培养3小时后用酶标仪在波长450nm处测定吸光度值(参考波长:630nm)。实验数据采用四参数回归计算法进行处理,分别计算出两种蛋白的EC50值,实验重复3次,两组差异统计分析采用t检验。
实施例1蛋白酶解及糖基化修饰共同作用致使PDGF-BBThr6多种单体的形成
我们首先怀疑蛋白酶解是造成多种PDGF-B单体形式的原因。通过SDS-PAGE还原电泳,分离PDGF-B的不同单体,考马斯亮蓝染色共检测出5条带(图3A)。将这5个蛋白条带分别进行N-末端氨基酸序列测序,结果显示,第1、2、3条带的N末端前5个氨基酸序列为TIAEP,为PDGF-BThr6正确N端序列,而第4、5条带的N端序列为TNANF。经蛋白序列比对,确定4、5号蛋白片段为Arg32-Thr33发生蛋白酶切而产生的截短型蛋白(图3A)。
但是这并不能解释形成至少5种PDGF单体的原因。第1、2、3条带和第4、5条带之间的分子量的差异也许是由于C末端酶切导致的。为了回答这个问题,我们利用一株特异的抗PDGF-B C末端的单抗(F-3)(Santa CruzBiotechnology,SC-365805)进行WB分析。检测结果表明,5条带中仅有2条带被检测出来。但是有趣的是,这两条与抗体结合的条带对应的似乎是第3和第5条蛋白片段(图3B)。如果第1、2、4条蛋白片段是由于发生了C末端酶切而导致不能被抗体检测到,那么它们的分子量应该更小,但是这显然与电泳检测的结果不符合。这意味着,还有其他原因有待进一步发现。
为了分析PDGF-B是否发生了糖基化修饰,我们利用肽N-糖苷酶F(PNGase F)对PDGF进行了酶切处理,并同时进行了SDS-PAGE电泳和糖蛋白染色分析。PNGaseF是一种酰胺酶,它几乎可以作用于糖肽/糖蛋白中所有的N-聚糖链,在糖链部分最内侧的GlcNAc和天冬酰胺残基之间进行切割,并将天冬酰胺变为天冬氨酸(10),是目前糖蛋白组学研究中鉴定N-糖蛋白应用最为广泛的一种酶。同为毕赤酵母表达的重组IFN-ω蛋白作用为N-糖基化蛋白的阳性对照。考马斯亮蓝染色结果表明,酶切前后PDGF-B蛋白的相对分子量没有发生变化,说明PDGF-B蛋白并没有发生N-糖基化(图3C,左)。然而糖蛋白染色的结果表明,PDGF-B确实是糖蛋白(图3C,右)。这意味着,毕赤酵母分泌表达的PDGF-B发生了O-糖基化修饰。同时,进一步分析发现,糖染色仅检测到了3条蛋白片段,应该分别对应SDS-PAGE结果中的第1、2、4号条带(图3B)。这也与上面WB的结果相吻合:第1、2、4蛋白片段在C-末端发生了糖基化,从而影响了PDGF-BThr6与抗体的结合。
综合上述实验结果,我们推断PDGF-BThr6多种形式的单体是由于第27/28位氨基酸Arg32-Thr33之间发生了蛋白酶解和蛋白翻译后不同程度的糖基化修饰共同作用造成的(表1)。
表1PDGF-BThr6修饰类型分析
注:表中完整PDGF是指PDGF-BThr6,即N端缺失5个氨基酸的PDGF-B。
实施例2PDGF-M1及PDGF-M2改构体的构建及蛋白性质检测
更进一步,我们想确认上述的推断,并希望使PDGF-B在毕赤酵母中的表达均一化。首先我们要确定可能的O-糖基化位点。利用在线网站CBS(www.CBS.dtu.dk)(11)对PDGF-BThr6蛋白序列进行糖基化位点预测,结果显示第6位、101位、109位的Thr为可能的O-糖基化修饰位点(图4)。相对于N-糖基化而言,O-糖基化位点并没有明确的基序,因而其预测也相对困难。但是,预测的第6、101、109位的潜在糖基化修饰位点与我们上述的结果是相符的:PDGF-BThr6蛋白的C末端应该有糖基化修饰(Thr101,Thr109),因为它们阻碍了与抗体的结合;在Thr33之前应该有糖基化修饰位点,因为这样才能解释发生酶解的PDGF-B条带中仅有一条(Band4)糖基化修饰的变体,而未发生酶解的糖基化PDGF-B单体有两条(Band1,2)(图3B;表1)。为了证实预测的结果,我们构建了两个PDGF-BThr6的突变体PDGF-M1及PDGF-M2。PDGF-M1突变了C端的两个糖基化位点,而PDGF-M2突变了全部3个潜在糖基化位点(模式图见图6A)。同时,为了去除Arg32-Thr33这一蛋白酶切位点,我们将Arg32突变为Pro,突变为了Pro是考虑到进化对氨基酸的选择。我们注意到成熟PDGF-B蛋白有60%的氨基酸序列与PDGF-A具有同衍生性,并且两者在结构与功能上也具有很高的相似性,而PDGF-A蛋白序列在对应位置的氨基酸序列正是Pro。
PDGF-M1的氨基酸序列为:
TIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVAAARPVA(SEQ ID NO:3);
核苷酸序列为:
ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCAGCTGCACGGCCTGTGGCC(SEQ ID NO:4)。
PDGF-M2的氨基酸序列为:
AIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVAAARPVA(SEQ ID NO:5);
核苷酸序列为:
GCCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCAGCTGCACGGCCTGTGGCC(SEQ ID NO:6)。
将编码PDGF-M1和PDGF-M2的DNA序列插入到pMEX9K表达载体,并整合入GS115毕赤酵母菌株中。通过甲醇诱导表达,并经色谱纯化。对纯化后PDGF-B蛋白进行了SDS-PAGE、糖蛋白染色及Western免疫印迹检测,以确定改构后蛋白性质。WB结果显示,两种突变体还原后仅能检测到单一条带,相对分子质量约为12kDa,与预期一致(图6B)。SDS-PAGE结果显示,PDGF-M2为单一条带,但是PDGF-M1在主带上仍有一微弱条带(图6C)。推测此带是由于Thr6的糖基化导致的。糖蛋白染色结果显示,相对于突变前,两种突变体的糖基化水平非常低,已很难用糖蛋白染色的方法检测到(图6D)。上述结果表明,将第32位R突变为P,去除了潜在的Kex2蛋白酶切位点,阻止了Thr33截短型PDGF-B单体的形成,而将Thr6、101、109三个糖基化修饰位点的突变也去除了蛋白在翻译后形成不同程度糖基化修饰,成功使得PDGF-B蛋白在毕赤酵母中的表达均一化。
实施例3PDGF-M2促细胞增殖活性检测
为了分析Thr6-Ala、Thr101-Ala、Thr109-Ala糖基化位点的突变和Arg32-Pro KEX酶切位点的突变会不会影响到PDGF-BB的生物学活性,我们利用WST-1法检测了PDGF-BThr6和PDGF-M2对Balb/c3T3细胞的增殖活性。结果显示,PDGF-BThr6的EC50为5.434±0.6475ng/ml,而PDGF-M2的EC50为3.492±0.4078ng/ml,经t检验,改构后蛋白活性高于改构前,P值为0.0117(图7)。
实施例4PDGF-M2Arg32突变对表达水平的影响
为了提升PDGF-M2的表达水平,我们根据毕赤酵母在蛋白表达时对密码子选择的偏好性,利用在线工具JAVA Condon Adaptation Tool对PDGF-M2(即PDGF-IM-P)进行密码子优化,优化后编码DNA序列如下:
编码PDGF-IM-P的DNA序列:
5’GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACCCAACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT-3’(SEQ ID NO:7)。
蛋白序列同PDGF-M2。
在密码子优化的基础上,将Arg32分别突变为Val(PDGF-IM-V,Val密码子采用GTT,)和Ile(PDGF-IM-I,Ile密码子采用ATC),与PDGF-M2的表达水平进行分析,以分析Arg32的突变对蛋白表达的影响。
PDGF-IM-V的核苷酸序列为:
GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACGTTACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT(SEQ ID NO:8)。
PDGF-IM-I的核苷酸序列为:
GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAGTTTTCGAAATCTCTAGAAGATTGATCGACATCACTAACGCTAACTTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCTGCTGCTAGACCAGTTGCT(SEQ ID NO:9)。
将编码PDGF-IM-P、PDGF-IM-V、PDGF-IM-I的DNA序列连接酶切位点、终止子等序列后克隆至pMEX9K表达载体,并整合至GS115表达菌株。经组氨酸缺陷MD平板筛选后,随机挑选9个克隆,在试管内进行甲醇诱导表达。培养上清经SDS-PAGE电泳分析,PDGF-IM-P蛋白表达量明显高于其他两株(图8A)。
通过G418对GS115/PDGF-IM-P、GS115/PDGF-IM-V、GS115/PDGF-IM-P进行了多拷贝插入子(Multiple Inserts)克隆的筛选。对分别在G418浓度为2.0mg/ml和4.0mg/ml平板上生长克隆进行了表达分析。结果仍然表明,PDGF-IM-P的平均表达水平高于其余两株(图8B)。这说明,Arg32位点的突变会影响PDGF在毕赤酵母中分泌表达水平,而将位点突变为Pro对表达相对有利。
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
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Claims (16)
1.血小板衍生生长因子B突变体,其氨基酸序列为SEQ ID NO:5所示的序列。
2.血小板衍生生长因子同源或异源二聚体,其由两个权利要求1的血小板衍生生长因子B突变体通过链内及链间二硫键结合而成,或者由一个权利要求1的血小板衍生生长因子B突变体与一个血小板衍生生长因子A通过链内或链间二硫键结合而成。
3.核酸分子,其编码权利要求1的血小板衍生生长因子B突变体。
4.权利要求3的核酸分子,其核苷酸序列为SEQ ID NO:6所示的序列。
5.载体,其含有权利要求3或4的核酸分子。
6.宿主细胞,其含有权利要求5的载体。
7.权利要求6的宿主细胞,其为真核细胞。
8.权利要求7的宿主细胞,其中所述真核细胞为酵母细胞、哺乳动物细胞或昆虫细胞。
9.权利要求7的宿主细胞,其为毕赤酵母细胞。
10.权利要求1的血小板衍生生长因子B突变体的制备方法,其包括取权利要求7-9任一项所述的宿主细胞进行培养、表达以及任选的纯化的步骤。
11.权利要求10的制备方法,其中所述表达为诱导表达。
12.血小板衍生生长因子B突变体的纯化方法,其包括取含有血小板衍生生长因子B突变体的培养上清或细胞裂解液依次进行疏水层析、离子交换层析和凝胶层析的步骤;其中所述血小板衍生生长因子B突变体为权利要求1的血小板衍生生长因子B突变体。
13.权利要求12的纯化方法,其选自以下1)-3)项中的一项或几项:
1)用于疏水层析的层析介质为Phenyl Sepharose 6Fast Flow;
2)用于离子交换层析的层析介质为Source 30S;
3)用于凝胶层析的层析介质为Hiload Superdex 75prep grad。
14.权利要求12或13的纯化方法,其中,
所述的疏水层析包括以下步骤:
(1)取含有血小板衍生生长因子B突变体的培养上清或细胞裂解液用调节缓冲液调节电导,所述调节缓冲液加入后的终体系为10-50mM磷酸缓冲液,0.8-1M(NH4)2SO4,pH 6.8-7.5;
(2)用平衡缓冲液平衡柱子,所述平衡缓冲液的配方为10-50mM磷酸缓冲液,0.8-1M(NH4)2SO4,pH 6.8-7.5;
(3)样品上柱后,用平衡缓冲液洗柱;
(4)用洗脱缓冲液洗脱,收集目的蛋白,所述洗脱缓冲液的配方为,10-50mM磷酸缓冲液,30%-50%乙二醇,pH 6.8-7.5;
所述离子交换层析包括以下步骤:
(1)用平衡缓冲液稀释疏水层析的洗脱峰至电导值为6mS/cm以下,所述平衡缓冲液的配方为,10-50mM磷酸缓冲液,pH 6.8-7.5;
(2)用平衡缓冲液平衡柱子;
(3)样品上柱后,用平衡缓冲液清洗柱子;
(4)用洗脱缓冲液梯度洗脱,收集目的蛋白,所述洗脱缓冲液的配方为10-50mM磷酸缓冲液,0.8-1.2M M NaCl,pH 6.8-7.5;
所述凝胶层析包括以下步骤:
(1)用磷酸盐缓冲液平衡柱子,所述磷酸盐缓冲液的配方为,10-50mM磷酸缓冲液,0.1-0.5M NaCl,pH 6.8-7.5;
(2)将离子交换层析的洗脱峰上样,每次上样体积不超过柱体积的0.3-4%;
(3)继续用步骤(1)中的磷酸盐缓冲液清洗柱子,收集目的蛋白,即得到纯化的血小板衍生生长因子B突变体。
15.权利要求1的血小板衍生生长因子B突变体用于制备促进细胞分裂、增殖、促进伤口愈合、皮肤再生、骨骼与牙齿缺损再生、关节修复的药物中的用途。
16.一种使血小板衍生生长因子表达更均一化的方法,其包括对野生型血小板衍生生长因子的氨基酸序列进行改造的步骤,所述改造包括:
a)将第101位和第109位氨基酸突变为丙氨酸;
b)将第6位氨基酸突变为丙氨酸;
c)将第32位氨基酸突变为脯氨酸;
所述改造还包括将所述氨基酸序列N端缺失5个氨基酸;
其中,所述野生型血小板衍生生长因子B的氨基酸序列N端前5个氨基酸依次为丝氨酸、亮氨酸、甘氨酸、丝氨酸、亮氨酸,并且缺失所述5个氨基酸后得到的序列如SEQ ID NO:1所示。
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