CN114874312A - 用于延长蛋白质半衰期的方法 - Google Patents
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Abstract
本发明涉及一种通过替换蛋白质一个或更多个氨基酸残基延长蛋白质或(多)肽半衰期的方法。进一步地,本发明关于通过上述方法制备的具有延长的半衰期的蛋白质。
Description
本申请是申请日为2016年10月30日,申请号为201680071485.0,发明名称为“用于延长蛋白质半衰期的方法”的分案申请。
技术领域
本发明涉及一种通过替换蛋白质的与泛素化相关的一个或更多个赖氨酸残基来延长蛋白质或(多)肽半衰期的方法,且涉及具有延长的半衰期的蛋白质。
背景技术
真核细胞中的蛋白质或(多)肽通过溶酶体系统和泛素-蛋白酶体系统两种不同途径被降解。分解10-20%的细胞蛋白的溶酶体系统,即没有底物特异性也没有精密时序可控性。也就是说,溶酶体系统是一种尤其分解大多数细胞外蛋白或膜蛋白的过程,例如表面蛋白通过内吞作用被细胞摄取,再被溶酶体降解。至于真核细胞中蛋白质的选择性降解,应涉及泛素-蛋白酶体途径(UPP),在这之中,目标蛋白首先结合泛素结合酶以形成多聚泛素链,再被蛋白酶体识别并分解。大约80-90%的真核细胞蛋白是通过UPP降解的,因此,UPP被认为调控真核细胞内大部分蛋白的降解,且负责体内的蛋白质转换和体内稳态。泛素是一个由76个高度保守的氨基酸组成的小蛋白,且它存在于所有真核细胞中。在泛素的所有氨基酸残基中,第6、11、27、29、33、48和63位残基是赖氨酸(Lysine,Lys,K),且已知第48位和63位的残基对多聚泛素链的形成起关键作用。通常称为E1、E2、E3的三种酶连续作用以促进泛素化,而被泛素标记的蛋白则被ATP依赖的蛋白质降解复合物中的26S蛋白酶体分解。
如上所述,泛素蛋白酶体途径(UPP)由两个分离且连续的过程组成。一个过程是一定数目的泛素分子连接至底物蛋白的蛋白标记过程,而另外一个则是经标记的蛋白被26S蛋白酶体复合物降解的过程。泛素和底物蛋白之间的结合是通过泛素C末端的甘氨酸残基和底物的赖氨酸残基之间形成异肽键,然后再通过一系列酶(泛素激活酶E1、泛素结合酶E2、泛素连接酶E3)在泛素和底物蛋白之间形成硫脂键而实施的。已知E1(泛素激活酶)通过ATP依赖的反应机制激活泛素。被激活的泛素被转移至E2(泛素结合酶)的泛素结合域中的半胱氨酸残基上,然后E2运送被激活的的泛素到E3连接酶处或直接运送至底物蛋白处。E3同样催化底物蛋白的赖氨酸残基和泛素的甘氨酸残基之间形成异肽键。另外一个泛素能够被连接至与底物蛋白相连的泛素的C末端的赖氨酸残基上,额外的泛素部分的重复连接通过一定数目的泛素分子彼此相连而产生多聚泛素链。当多聚泛素链被生成之后,底物蛋白则被26S蛋白酶体选择性地识别并降解。
与此同时,生物体内有着多种具有治疗效果的蛋白质。这些在体内具有治疗效果的蛋白质或(多)肽或生物活性多肽包括,但不限于,例如,生长激素释放激素(GHRH)、生长激素释放肽、干扰素(干扰素-α或干扰素-β)、干扰素受体、集落刺激因子(CSF)、胰高血糖素样肽、白介素、白介素受体、酵素、白介素结合蛋白、细胞因子结合蛋白、G蛋白偶联受体、人生长激素(hGH)、巨噬细胞活化因子、巨噬细胞肽、B细胞因子、T细胞因子、蛋白A、过敏抑制剂、细胞坏死糖蛋白、G蛋白偶联受体、免疫毒素、淋巴毒素、肿瘤坏死因子、肿瘤抑制因子、转移生长因子、α-1抗胰蛋白酶、白蛋白、α-乳白蛋白、载脂蛋白E、红细胞生成素、高度糖基化红细胞生成素、血管生成素、血红蛋白、凝血酶、凝血酶受体激活肽、血栓调节蛋白、第七因子、第七因子a、第八因子、第九因子、第十三因子、纤维蛋白溶酶原激活因子、尿激酶、链激酶、水蛭素、蛋白C、C反应蛋白、肾素抑制剂、胶原蛋白酶抑制剂、过氧化物歧化酶、瘦蛋白、血小板源生长因子、上皮生长因子、表皮生长因子、血管抑素、血管紧张肽、骨生长因子、骨刺激蛋白、降血钙素、胰岛素、心钠素、软骨诱导因子、纤维蛋白结合肽、依降钙素、结缔组织激活因子、组织因子通道抑制剂、促卵泡激素、促黄体激素、促黄体激素释放激素、神经生长因子、甲状旁腺素、松弛肽、分泌素、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、肠促胰酶肽、胰多肽、胃泌素释放肽、促皮质素释放因子、促甲状腺激素、自毒素、乳铁蛋白、肌生成抑制蛋白、受体、受体拮抗体、细胞表面抗原、病毒衍生的疫苗抗原、单克隆抗体、多克隆抗体和抗体片段。
粒细胞集落刺激因子(G-CSF),一种糖蛋白,其产生骨髓干细胞和粒细胞,且刺激骨髓分泌骨髓干细胞和粒细胞到血管中。G-CSF是一种集落刺激因子,并且也行使激素和细胞因子的功能。进一步地,C-CSF还通过增加神经可塑性和抑制细胞凋亡而作为一种神经营养因子,除此之外还对造血作用产生影响。G-CSF受体在脑和脊柱中的神经元中被表达。在中枢神经系统中,G-CSF诱导神经元的生成和增加神经可塑性,从而与细胞凋亡相关。因此,G-CSF被研究用于治疗神经元疾病,例如脑梗死。G-CSF可以刺激粒细胞的生成,其是白细胞的一种。进一步地,重组G-CSF可以被用于加快在肿瘤学和血液学中的化学治疗引起的嗜中性白细胞减少症的恢复。研究报道称G-CSF激活神经胶质瘤细胞中的STAT3从而参与胶质瘤的生长(Cancer Biol Ther.,13(6),389-400,2012)。进一步地,据报道,G-CSF在卵巢上皮癌细胞中被表达,且通过调节JAK2/STAT3通路与女性子宫癌病理性相关(Br J Cancer,110,133-145,2014)。
与体内稳态有关的蛋白质治疗剂具有各种各样的副作用,诸如增加癌症诱发的风险。例如,已知肠促胰岛素降解酶(DPP-4)(二肽基肽酶-4)抑制剂家族治疗剂会增加甲状腺癌的诱发的可能,而已知甘精胰岛素会增加乳腺癌的风险。进一步地,向遭受生长激素分泌紊乱的疾病的病人持续性或过量性的施用生长激素会被卷入糖尿病、微血管疾病和病人的过早死亡。就这一点而言,有广泛的研究来减少治疗性蛋白的不利和副作用。延长蛋白的半衰期被认为是减少治疗性蛋白的不利和副作用风险的一种方法。为此,大量的方法已经被公开。就这一点,本发明人已经研究开发一种用于延长体内和/或体外蛋白的半衰期的新方法,并通过替换治疗性蛋白或(多)肽中与泛素化相关的一个或更多个赖氨酸残基以阻止蛋白或者(多)肽通过泛素-蛋白酶体系统降解而完成本发明。
本文所引用的全部专利、出版物以及参考文献的教导通过引用的方式全部并入本申请中。
发明内容
技术问题
本发明的目的是增加蛋白质或(多)肽的半衰期。
进一步地,本发明的另一个目的是提供一种具有延长半衰期的治疗性蛋白。
进一步地,本发明的另一个目的是提供一种药物组合物,包括作为药物活性成分的具有延长半衰期的蛋白。
解决问题的方案
为达到目的,本发明提供一种通过替换蛋白质的氨基酸的一个或多个赖氨酸残基而在体内和/或体外延长蛋白质半衰期的方法。
在本发明中,赖氨酸残基能够被保守性氨基酸替换。术语“保守性氨基酸替换”是指一个氨基酸被另外一个与该氨基酸不同但是具有相似的化学性质的氨基酸替换,例如电荷和疏水性。一般说来,使用相应地保守性氨基酸的氨基酸替换,一个蛋白质的功能特点不会被本质地改变。例如,氨基酸根据侧链具有的相似的化学性质能够被分类,如下:①脂肪族侧链:甘氨酸、丙氨酸、缬氨酸、亮氨酸、和异亮氨酸;②脂肪族-羟基侧链:丝氨酸、苏氨酸;③酰胺侧链:天冬酰胺和谷氨酰胺;④芳香族侧链:苯丙氨酸、络氨酸、色氨酸;⑤碱性侧链:赖氨酸、精氨酸和组氨酸;⑥酸性侧链:天冬氨酸和谷氨酸;和⑦含硫侧链:半胱氨酸和甲硫氨酸。
在本发明中,赖氨酸残基能够被具有碱性侧链的精氨酸和组氨酸替换。优选为,赖氨酸残基被精氨酸替换。
本发明的有益效果
根据本发明,被精氨酸替换一个或者多个赖氨酸残基的变体蛋白质具有显著延长的半衰期,因此能够维持一段很长的时间。
附图说明
图1为BMP2表达载体的结构;
图2为BMP2基因PCR克隆产物的结果;
图3为HEK-293T细胞中BMP2质粒基因的表达;
图4为通过泛素化实验解释BMP2的蛋白质水解途径;
图5为相比野生型,用精氨酸替换赖氨酸残基的经取代的BMP2的泛素化水平;
图6为经过蛋白合成抑制剂环十二碳三烯(CHX)处理之后的BMP2的半衰期改变;
图7为JAK-STAT信号转导样效应的结果。
在下文中,将结合实施例对本发明进行更为详细的描述。应该明白的是这些实施例不能以任何方式被考虑为限定本发明。
具体实施方式
在本发明的一个实施方案中,所述蛋白是骨形态生成蛋白-2(BMP-2)。在BMP-2的氨基酸序列中(SEQNo.52),对应于N端第32、64、127、178、185、236、241、272、278、281、285、287、290、293、297、355、358、379和383位的至少有一个赖氨酸被替换为精氨酸。因此,提供了具有增加的半衰期的BMP-2。进一步地,提供了包含经取代的BMP-2的药物组合物以预防和/或治疗品贫血和骨病(Cell J.,17(2),193-200,2015;Clin Orthop Relat Res.,318,222-230,1995)。
在本发明中,采用定点突变的方法将蛋白质氨基酸序列中的赖氨酸残基替换为精氨酸(R)。根据该方法,使用DNA序列设计引物而诱导定点突变,然后在特定条件下通过执行PCR而产生突变的质粒DNA。
在本发明中,通过向细胞系转染目标蛋白和使用免疫沉淀反应测量泛素化程度。如果在使用MG132处理之后的被转染细胞系中泛素化水平升高,这就说明目标蛋白通过泛素-蛋白酶体途径被降解。
本发明的药物组合物可以通过多种方式被施用进入身体,包括口服,经皮肤,经皮下,静脉内或肌肉施用,而更优选方案则是通过注射的形式制备。进一步地,本发明的药物组合物还可以通过本领域技术人员熟知的方法而被制作而在施用后提供活性物质的快速,保持或延迟地释放。制剂的形式可以为片剂、丸剂、粉剂、小袋、酏剂、悬浮剂、乳剂、溶液、糖浆、气雾剂、软和硬的胶囊、无菌注射液、无菌压缩粉末等。合适的载体、赋形剂和稀释剂的例子是乳糖、葡萄糖、蔗糖、甘露醇、木糖醇、赤藓醇、麦芽糖醇、淀粉、阿拉伯树胶、海藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油。进一步地,药剂可以另外地包括填充料、抗粘剂、润滑剂、湿润剂、芳香剂、乳化剂、防腐剂等。
合适的载体、赋形剂、稀释剂的例子是乳糖、葡萄糖、蔗糖、甘露醇、木糖醇、赤藓醇、麦芽糖醇、淀粉、阿拉伯树胶、海藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、滑石、硬脂酸镁、矿物油。进一步的,药剂可以另外地包括填充料、抗粘剂、润滑剂、湿润剂、芳香剂、乳化剂、防腐剂等。
在此文中使用的,单数形式“a”,“an”,“the”也意图包括复数形式,除非本文中清楚地表明其他含义。此外,说明书和/或权利要求中使用的术语“包括(including)”,“包括(includes)”,“具有(having)”,“具有(has)”,“和(with)”,“例如”或它们的变体形式,并不起限定作用,也旨在是包括的方式类似于术语“包括(comprising)”。在本发明中,“生物活性的多肽或蛋白”是当被施用给哺乳动物包括人类时,表现出有用的生物学活性的(多)肽或蛋白。
发明方式
下述实施例提供了说明性的实施方案。根据本发明公开的内容和本领域技术人员的通常水平,本领域技术人员应理解下述实施例仅意图作为示例,且不脱离本发明请求保护的主题的范围能够做出很多改变、修饰和变化。
实施例1骨形态生成蛋白2(BMP2)泛素化和半衰期延长的分析,以及细胞中的信号转导的分析
1.骨形态生成蛋白2(BMP2)表达载体克隆和蛋白表达
(1)骨形态生成蛋白2(BMP2)表达载体克隆
通过PCR扩增的骨形态生成蛋白2(BMP2)DNA被EcoRI和XhoI处理,再连接到预先以同样的酶进行消化的pcDNA3-myc(5.6kb)载体上(图1,BMP2氨基酸序列:SEQ No.52)。于是,在克隆载体的限制性内切酶消化之后,通过琼脂糖凝胶电泳确认DNA插入片段的存在(图2)。图1中下划线标黑的核苷酸序列表明用于PCR并确定克隆位点的引物设定(图2)。PCR的条件如下所示:第一步:94℃3分钟(1次循环);第二步:94℃30秒;58℃30秒;72℃1分钟30秒(25次循环);第三步:72℃10分钟(1次循环),之后再保存于4℃。为了克隆DNA编码的蛋白表达的评估,利用抗-myc抗体(9E10,sc-40)与图1图谱的pcDNA3-myc载体上的myc的结合而进行蛋白质印迹法。蛋白质印记的结果显示结合myc的BMP2被很好地表达。肌动蛋白作为标准确保蛋白质上样量的是合适的(图3)。
(2)赖氨酸(Lysine,K)残基的取代
使用定点突变的方法将赖氨酸残基替换为精氨酸(Arginine,R)。下述引物设定被用于PCR以制备经取代的质粒DNA。
(BMP2 K293R)FP 5'-GAAACGCCTTAGGTCCAGCTGTAAGAGAC-3'(SEQ No.53),RP 5'-GTCTCTTACAGCTGGACCTAAGGCGTTTC 3'(SEQ No.54);
(BMP2 K297R)FP 5'-TTAAGTCCAGCTGTAGGAGACACCCTTTGT-3'(SEQ No.55),RP 5'-ACAAAGG GTGTCTCCTACAGCTGGACTTAA-3'(SEQ No.56);
(BMP2 K355R)FP 5'-GTTAACTCTAGGATTCCTAAGGC-3'(SEQ No.57),RP 5'-GCCTTAGGAATCCTAGAGTTAAC-3'(SEQ No.58);and
(BMP2 K383R)FP 5'-GGTTGTATTAAGGAACTATCAGGAC-3'(SEQ No.59),RP 5'-GTCCTGATAGTTCCTTAATACAACC-3'(SEQ No.60)
通过使用pcDNA3-myc-BMP2作为模板,制备有一个或更多个赖氨酸被替换为精氨酸(K→R)的5种质粒DNA。
表1
赖氨酸(K)残基 | 构建的BMP2,精氨酸替换赖氨酸 |
293 | pcDNA3-myc-BMP2(K293R) |
297 | pcDNA3-myc-BMP2(K297R) |
355 | pcDNA3-myc-BMP2(K355R) |
383 | pcDNA3-myc-BMP2(K383R) |
2.体内泛素化分析
HEK293T细胞被转染编码pcDNA3-myc-BMP2 WT和pMT123-HA-ubiquitin的质粒。为了泛素化水平的分析,pcDNA3-myc-BMP2 WT 2μg和pMT123-HA-ubiquitin 1μgDNA被共转入细胞中。转染后24小时,再对细胞进行MG132(蛋白酶体抑制剂,5μg/ml)处理6小时,再进行免疫沉淀反应分析(图4)。于是,HEK293T细胞被分别转入编码pcDNA3-myc-BMP2 WT,pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R),pcDNA3-myc-BMP2变体(K383R)和pMT123-HA-ubiquitin的质粒。为了泛素化水平的分析,pMT123-HA-ubiquitin 1μg分别与2μg pcDNA3-myc-BMP2 WT,pcDNA3-myc-BMP2变体(K62R),pcDNA3-myc-BMP2变体(K124R),pcDNA3-myc-BMP2变体(K153R)和pcDNA3-myc-BMP2变体(K158R)共转入细胞中。接下来,转染后24小时,进行免疫沉淀反应(图5)。通过免疫沉淀反应获得的蛋白样品被溶于缓冲液中,其包含(1%Triton X,150mM NaCl,50mMTris-HCl,pH 8和1mM PMSF(苯甲基硫磺酰)),再将其与抗myc(9E10)一抗(Santa CruzBiotechnology,sc-40)混合。此后,混合物被孵育在4℃并过夜。免疫沉淀物可以通过与A/G珠在4℃反应2小时而分离。随后,分离的免疫沉淀物用缓冲液清洗两次。在与2X的SDS缓冲液混合并加热至100℃,7分钟之后,蛋白样品通过SDS凝胶电泳分离。分离的蛋白被转移到聚偏氟乙烯(PVDF)膜上,再使用以1:1000稀释的抗小鼠的二抗(过氧化物酶标记的小鼠IgG(H+L),KPL,074-1806)和包含有抗myc抗体(9E10,sc-40),抗HA(sc-7392)和抗β-actin抗体(sc-47778)封闭液通过ECL系统进行显影。结果,当使用抗myc抗体(9E10,sc-40)进行免疫沉淀反应时,多聚泛素链可以通过泛素与pcDNA3-myc-BMP2 WT的结合而形成,因此强烈的代表泛素存在的条带被检测到(图4,泳道3和4)。进一步地,当细胞被MG132处理六小时时,多聚泛素链的形成增加,因此出现更加强烈的代表泛素的条带(图4,泳道4)。进一步地,至于pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R)和pcDNA3-myc-BMP2变体(K355R),条带不如野生型的强烈,且更少量的泛素被检测到,因为pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R)不能与泛素相结合(图5,泳道3-5)。这些结果说明BMP2先结合泛素,再通过形成的多聚泛素链被泛素-蛋白酶体系统降解。
3使用蛋白酶合成抑制剂环十二碳三烯(CHX)评估BMP2的半衰期
HEK293T细胞被分别的转入2μg pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R)和pcDNA3-myc-BMP2变体(K383R)。转染48小时后,细胞用蛋白酶合成抑制剂环十二碳三烯(CHX)(Sigma-Aldrich,100μg/ml)处理,在抑制剂处理之后的4小时和8小时,分别检测每种蛋白的半衰期。结果,观察人BMP2的降解(图6)。人BMP2的半衰期少于2小时,而pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R)的半衰期被延长至4小时或者更多,如图6所示。
4.BMP2和经取代的BMP2在细胞中的信号转导
已经知道在多种骨髓瘤细胞中,骨形态生成蛋白可以抑制STAT3,因此诱导细胞凋亡(Blood,96,2005-2011,2000)。在这个实验中,我们检测了BMP2和经取代的BMP2在细胞中的信号转导。首先,HepG2细胞饥饿8小时,再分别转入3μg pcDNA3-myc-BMP2 WT,pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R)和pcDNA3-myc-BMP2变体(K383R)。转染2天后,细胞内的蛋白被提取并定量。蛋白质印记法被用于分析细胞中的信号转导。从分别转染pcDNA3-myc-BMP2 WT,pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R)和pcDNA3-myc-BMP2变体(K383R)的HepG2细胞中分离得到的蛋白被移至聚偏氟乙烯(PVDF)膜上。于是,使用以1:1000稀释的抗兔和抗鼠的二抗和包含有抗-STAT3(sc-21876),抗磷酸化STAT3(Y705,cell signaling 9131S)和抗β-actin抗体(sc-47778)封闭液通过ECL系统对蛋白质进行显影。结果,相比于野生型,pcDNA3-myc-BMP2变体(K293R),pcDNA3-myc-BMP2变体(K297R),pcDNA3-myc-BMP2变体(K355R),pcDNA3-myc-BMP2变体(K383R)在HepG2细胞中,显示出一样或者增加的磷酸化STAT3信号转导(图7)。
实用性
因为本发明的变体蛋白具有延长的半衰期,本发明可以被用于开发蛋白或者(多)肽治疗性试剂。
序列表
<110> UBI蛋白公司
<120> 用于延长蛋白质半衰期的方法
<130> UBPRN16P01WO
<150> KR 10-2015-0160728
<151> 2015-11-16
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Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu
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Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu
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Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg
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Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser
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His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe
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Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys
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Arg Ser Val Glu Gly Ser Cys Gly Phe
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Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu
1 5 10 15
Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly
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Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe
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Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly
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Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu
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<212> PRT
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Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys
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Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu
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Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser
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Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu
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Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
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Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
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Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val
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Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys
130 135 140
Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro
145 150 155 160
Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
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Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
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<212> DNA
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<220>
<223> 人干扰素α
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<212> PRT
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Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln
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Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro
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Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu
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Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
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Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
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Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
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Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
100 105 110
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
130 135 140
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
145 150 155 160
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
165 170 175
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
180 185 190
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
195 200 205
<210> 32
<211> 24
<212> DNA
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<220>
<223> 人 G-CSF
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<210> 33
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<212> DNA
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<212> DNA
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<220>
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<212> DNA
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<220>
<223> 人 G-CSF
<400> 35
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<210> 36
<211> 187
<212> PRT
<213> 人工序列
<220>
<223> 人干扰素β
<400> 36
Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser
1 5 10 15
Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg
20 25 30
Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
35 40 45
Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu
50 55 60
Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile
65 70 75 80
Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser
85 90 95
Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val
100 105 110
Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu
115 120 125
Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys
130 135 140
Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser
145 150 155 160
His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr
165 170 175
Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn
180 185
<210> 37
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 37
cagtgtcaga ggctcctgtg g 21
<210> 38
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 38
ccacaggagc ctctgacact g 21
<210> 39
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 39
ctggaagaaa gactggagaa a 21
<210> 40
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 40
tttctccagt ctttcttcca g 21
<210> 41
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 41
cattacctga gggccaagga g 21
<210> 42
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人干扰素β
<400> 42
ctccttggcc ctcaggtaat g 21
<210> 43
<211> 193
<212> PRT
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 43
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu
1 5 10 15
Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu
20 25 30
Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu
35 40 45
Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu
50 55 60
Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg
65 70 75 80
Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu
85 90 95
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
100 105 110
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly
115 120 125
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu
130 135 140
Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile
145 150 155 160
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu
165 170 175
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp
180 185 190
Arg
<210> 44
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 44
gcatgtggat agagccgtca gtgc 24
<210> 45
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 45
gcactgacgg ctctatccac atgc 24
<210> 46
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 46
tgacactttc cgcagactct tccgagtcta c 31
<210> 47
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 47
gtagactcgg aagagtctgc ggaaagtgtc a 31
<210> 48
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 48
ctccggggaa ggctgaagct g 21
<210> 49
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 49
cagcttcagc cttccccgga g 21
<210> 50
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 50
ggaaagctga ggctgtacac agg 23
<210> 51
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人红细胞生成素
<400> 51
cctgtgtaca gcctcagctt tcc 23
<210> 52
<211> 396
<212> PRT
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 52
Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val
1 5 10 15
Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys
20 25 30
Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu
35 40 45
Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys
50 55 60
Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu
65 70 75 80
Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp
85 90 95
His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe
100 105 110
His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr
115 120 125
Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe
130 135 140
Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala
145 150 155 160
Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile
165 170 175
Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu
180 185 190
Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp
195 200 205
Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His
210 215 220
Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser
225 230 235 240
Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser
245 250 255
Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys
260 265 270
Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln
275 280 285
Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp
290 295 300
Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr
305 310 315 320
His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His
325 330 335
Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val
340 345 350
Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala
355 360 365
Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn
370 375 380
Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg
385 390 395
<210> 53
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 53
gaaacgcctt aggtccagct gtaagagac
29
<210> 54
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 54
gtctcttaca gctggaccta aggcgtttc
29
<210> 55
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 55
ttaagtccag ctgtaggaga caccctttgt
30
<210> 56
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 56
acaaagggtg tctcctacag ctggacttaa
30
<210> 57
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 57
gttaactcta ggattcctaa ggc
23
<210> 58
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 58
gccttaggaa tcctagagtt aac
23
<210> 59
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 59
ggttgtatta aggaactatc aggac
25
<210> 60
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 人骨形态生成蛋白-2
<400> 60
gtcctgatag ttccttaata caacc
25
<210> 61
<211> 155
<212> PRT
<213> 人工序列
<220>
<223> 人纤维母细胞生长因子-1
<400> 61
Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe
1 5 10 15
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser
20 25 30
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly
35 40 45
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu
50 55 60
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
65 70 75 80
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu
85 90 95
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr
100 105 110
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
115 120 125
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala
130 135 140
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
145 150 155
<210> 62
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人纤维母细胞生长因子-1
<400> 62
aagaagccca gactcctcta c 21
<210> 63
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人纤维母细胞生长因子-1
<400> 63
gtagaggagt ctgggcttct t 21
<210> 64
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人纤维母细胞生长因子-1
<400> 64
catgcagaga ggaattggtt t 21
<210> 65
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人纤维母细胞生长因子-1
<400> 65
aaaccaattc ctctctgcat g 21
<210> 66
<211> 167
<212> PRT
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 66
Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu
1 5 10 15
Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys
20 25 30
Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr
35 40 45
Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro
50 55 60
Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala
65 70 75 80
Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln
85 90 95
Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala
100 105 110
Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu
115 120 125
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
130 135 140
Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln
145 150 155 160
Leu Asp Leu Ser Pro Gly Cys
165
<210> 67
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 67
cccatccaaa aggtccaaga t 21
<210> 68
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 68
atcttggacc ttttggatgg g 21
<210> 69
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 69
gatgacacca agaccctcat c 21
<210> 70
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 70
gatgagggtc ttggtgtcat c 21
<210> 71
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 71
accctcatca ggacaattgt c 21
<210> 72
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 72
gacaattgtc ctgatgaggg t 21
<210> 73
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 73
accttatcca ggatggacca g 21
<210> 74
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人瘦蛋白
<400> 74
ctggtccatc ctggataagg t 21
<210> 75
<211> 209
<212> PRT
<213> 人工序列
<220>
<223> 人血管内皮生长因子A
<400> 75
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu
1 5 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30
Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln
35 40 45
Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu
50 55 60
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu
65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro
85 90 95
Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
100 105 110
Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys
115 120 125
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val
130 135 140
Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Pro
145 150 155 160
Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro
165 170 175
Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala
180 185 190
Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg
195 200 205
Arg
<210> 76
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 人血管内皮生长因子A
<400> 76
tacagcacaa cagatgtgaa tgcagacc 28
<210> 77
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 人血管内皮生长因子A
<400> 77
ggtctgcatt cacatctgtt gtgctgta 28
<210> 78
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 人血管内皮生长因子A
<400> 78
atccgcagac gtgtagatgt tcctgca 27
<210> 79
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 人血管内皮生长因子A
<400> 79
tgcaggaaca tctacacgtc tgcggat 27
<210> 80
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> 人prepro-GHRL
<400> 80
Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu
1 5 10 15
Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His
20 25 30
Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu
35 40 45
Gln Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln
50 55 60
Ala Glu Gly Ala Glu Asp Glu Met Glu Val Arg Phe Asn Ala Pro Phe
65 70 75 80
Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gln His Ser Gln
85 90 95
Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu
100 105 110
Ala Pro Ala Asp Lys
115
<210> 81
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人prepro-GHRL
<400> 81
gccctgggga ggtttcttca g 21
<210> 82
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 人prepro-GHRL
<400> 82
ctgaagaaac ctccccaggg c 21
<210> 83
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 83
Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys
1 5 10 15
Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg
20 25
<210> 84
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 84
agtccagcag agaagggagt cgaagaagcc a 31
<210> 85
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 85
tggcttcttc gactcccttc tctgctggac t 31
<210> 86
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 86
agaaaggagt cgaggaagcc accagccaag c
31
<210> 87
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 87
gcttggctgg tggcttcctc gactcctttc t
31
<210> 88
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 88
agaaaggagt cgaagaggcc accagccaag c
31
<210> 89
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 89
gcttggctgg tggcctcttc gactcctttc t
31
<210> 90
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 90
aagaagccac cagccaggct gcagccccga
30
<210> 91
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人食欲刺激激素(胃饥饿素)
<400> 91
tcggggctgc agcctggctg gtggcttctt
30
<210> 92
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 人胰高血糖素样肽-1(GLP-1)
<400> 92
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 93
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 人胰高血糖素样肽-1(GLP-1)
<400> 93
aagctgccag ggaattca 18
<210> 94
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 人胰高血糖素样肽-1(GLP-1)
<400> 94
tgaattccct ggcagctt 18
<210> 95
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> 人胰高血糖素样肽-1(GLP-1)
<400> 95
ttggctggtg agaggcc 17
<210> 96
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> 人胰高血糖素样肽-1(GLP-1)
<400> 96
ggcctctcac cagccaa 17
<210> 97
<211> 470
<212> PRT
<213> 人工序列
<220>
<223> 人IgG重链
<400> 97
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
1 5 10 15
Val Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
85 90 95
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Leu Glu
465 470
<210> 98
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 98
acaaaggtgg acaggaaggt ggagcccaag 30
<210> 99
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 99
cttgggctcc accttcctgt ccacctttgt 30
<210> 100
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 100
gagtataagt gcagggtgtc caataaggcc ctgc 34
<210> 101
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 101
gcagggcctt attggacacc ctgcacttat actc 34
<210> 102
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 102
ctttctgtat agcaggctga ccgtggataa gtcc 34
<210> 103
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 人IgG重链
<400> 103
ggacttatcc acggtcagcc tgctatacag aaag 34
<210> 104
<211> 238
<212> PRT
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 104
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
210 215 220
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Leu Glu
225 230 235
<210> 105
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 105
cctggcaagg ccccaaggct gctgatctac 30
<210> 106
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 106
gtagatcagc agccttgggg ccttgccagg 30
<210> 107
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 107
acaaaggtgg agatcaggag gaccgtggcc 30
<210> 108
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 108
ggccacggtc ctcctgatct ccacctttgt 30
<210> 109
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 109
gccaaggtgc agtggagggt ggataacgcc 30
<210> 110
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 人IgG轻链
<400> 110
ggcgttatcc accctccact gcaccttggc 30
Claims (4)
1.一种具有延长的半衰期的BMP2,其中,所述BMP2具有如SEQ No.51所示的氨基酸序列,且对应于所述BMP2的N末端第32、64、127、178、185、236、241、272、278、281、285、287、290、293、297、355、358、379和383位的一个或更多个赖氨酸残基被精氨酸替换。
2.一种用于预防和/或治疗贫血和骨病的药物组合物,其中,所述药物组合物包括权利要求1所述的BMP2以及药学上可接受的赋形剂。
3.一种表达载体,所述表达载体包含:(a)启动子;(b)编码权利要求1所述的BMP2的核苷酸序列;和任选的连接子,其中所述启动子和核苷酸序列可操作地连接。
4.一种宿主细胞,所述宿主细胞包含权利要求3所述的表达载体。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2015-0160728 | 2015-11-16 | ||
KR20150160728 | 2015-11-16 | ||
CN201680071485.0A CN108699120B (zh) | 2015-11-16 | 2016-10-30 | 用于延长蛋白质半衰期的方法 |
PCT/KR2016/012334 WO2017086627A1 (en) | 2015-11-16 | 2016-10-30 | A method for extending half-life of a protein |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201680071485.0A Division CN108699120B (zh) | 2015-11-16 | 2016-10-30 | 用于延长蛋白质半衰期的方法 |
Publications (1)
Publication Number | Publication Date |
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DK3892628T3 (da) | 2018-06-29 | 2022-11-14 | Akston Biosciences Corp | Ultra-langtidsvirkende insulin-fc-fusionsproteiner og fremgangsmåder til anvendelse |
US11267862B2 (en) | 2018-06-29 | 2022-03-08 | Akston Biosciences Corporation | Ultra-long acting insulin-Fc fusion proteins and methods of use |
WO2020118239A1 (en) * | 2018-12-06 | 2020-06-11 | Arcturus Therapeutics, Inc. | Modified proteins and associated methods of treatment |
CN110403904A (zh) * | 2019-07-26 | 2019-11-05 | 翔宇药业股份有限公司 | 卡贝缩宫素注射液及其应用 |
AU2020407365B2 (en) | 2019-12-19 | 2023-09-21 | Akston Biosciences Corporation | Ultra-long acting insulin-Fc fusion proteins and methods of use |
US11186623B2 (en) | 2019-12-24 | 2021-11-30 | Akston Bioscience Corporation | Ultra-long acting insulin-Fc fusion proteins and methods of use |
US11192930B2 (en) | 2020-04-10 | 2021-12-07 | Askton Bioscences Corporation | Ultra-long acting insulin-Fc fusion protein and methods of use |
LT3972987T (lt) | 2020-04-10 | 2023-09-25 | Akston Biosciences Corporation | Antigenui specifinė imunoterapija, skirta sulietiems covid-19 baltymams ir naudojimo būdai |
US11198719B2 (en) | 2020-04-29 | 2021-12-14 | Akston Biosciences Corporation | Ultra-long acting insulin-Fc fusion protein and methods of use |
CN113845583B (zh) * | 2020-06-28 | 2023-08-11 | 江苏中新医药有限公司 | 一种修饰的重组人神经生长因子及其制备方法 |
CN114685643A (zh) * | 2020-12-29 | 2022-07-01 | 苏州康宁杰瑞生物科技有限公司 | 一种人glp-1多肽变体及其应用 |
WO2023004406A2 (en) | 2021-07-23 | 2023-01-26 | Akston Biosciences Corporation | Insulin-fc fusion proteins and methods of use to treat cancer |
JP2024063927A (ja) | 2022-10-27 | 2024-05-14 | セイコーエプソン株式会社 | 印刷装置 |
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