CN112661858A - 重组人生长激素Fc融合蛋白和用途及其工程细胞株 - Google Patents
重组人生长激素Fc融合蛋白和用途及其工程细胞株 Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
本发明涉及生物医药工程技术领域,特别是基因重组长效人生长激素技术领域,具体涉及一种重组人生长激素Fc融合蛋白研制。本发明的人生长激素Fc融合蛋白所述融合蛋白含有人生长激素(hGH)、柔性肽接头(L))和人免疫球蛋白Fc片段。本发明还公开了该融合蛋白的活性测定方法以及能够表达该融合蛋白的宿主细胞。本发明构建的重组人生长激素Fc融合蛋白比重组hGH具有更长的血清半衰期,大大降低了治疗时间内所需的注射给药频率,从而提高患者的依从性、增加医患双方的便利性,同时其生产过程也更简单、高效,特别适用于工业化大规模制备基因重组长效人生长激素。
Description
技术领域
本发明涉及生物医药工程技术领域,特别是基因重组长效人生长激素技术领域,具体涉及一种重组人生长激素Fc融合蛋白研制和用途及其工程细胞株。
背景技术
生长激素(HGH)是一种肽类激素,它是由垂体中的生长激素细胞合成、存储和分泌。成熟野生型人GH(由SEQ ID NO:2鉴别的hGH)是由191个氨基酸残基构成,其中大部分是以分子量22KDa形式存在,少量生长激素以20KDa形式存在,等电点为4.9,在53、165、182和189位置含有4个半胱氨酸残基,其通过形成分别连接C53与C165和C182与C189的两个分子内二硫键,稳定蛋白的三维结构。生长激素与其特异生长激素受体(hGHR)在靶点细胞表面相互结合,介导出生化串联反应,活化细胞内的JAK2-STATs、MAPK和PI3K-GSK3等多条明确的信号通路,从而维持细胞的增殖和分化。胰岛素样生长因-1(IGF-1)与生长激素受体组合表达,参与细胞的生长和再生。众所周知,生长激素是在正常人体的脑垂体中产生的,并逐渐增加到***,并随着年龄逐渐减少。
生长激素最主要的生理作用是通过促进身体各组织、器官以及骨骼中受体细胞的蛋白质合成和脂肪的分解,进而促进人的代谢和生长发育。如果人体的生长激素分泌不足,通俗的说就是会导致“发育不良”。因此,目前临床上,生长激素主要被用于治疗儿童生长迟缓和成人的生长激素不足。此外,近年来在美容和抗衰老以及重度烧伤等方面也有了应用,经实验证实生长激素确实能够使体内脂肪重新分泌,会让皮下弹性组织中的粘多糖增加,使皮肤变得坚实有弹性。
2019年国内生长激素存量市场约55亿元,处于快速增长期。根据出厂口径计算,生长激素市场规模逐年升高,从2014年11.90亿元逐渐增长到2019年55.19亿,复合增长率35.91%。目前国内外重组人生长激素已有多个产品上市,例如辉瑞的健豪宁(注射用重组人生长激素)其适应症为:由内源性生长激素分泌不足所致或由Turner's综合征或慢性肾功能不全引起的生长障碍;Genentech的NUTROPIN;诺和诺德的(somatropin,生长激素),金赛药业的赛增(重组人生长激素注射液)以及安科生物的安苏萌(注射用重组人生长激素)等。
然而,这些用药多为短效产品,在临床应用时,由于生长激素在人体内半衰期很短,半衰期大约2小时,所以治疗用药需要皮下注射每天一针,而且给药周期长(例如可高达约6个月以上)。频繁的注射生长激素会给患者带来不便,用药成本也比较昂贵。而市场开发的长效的PEG(聚乙二醇)化生长激素通过聚乙二醇修饰后虽然能够明显提高药物半衰期使其长效化,但由于PEG作为人体内异物有潜在风险,在注射的过程中可能引起疼痛感,安全性收到限制,而且PEG化生长激素的代谢途经肾小球滤过清除,有研究表明PEG成分与肾小管上皮细胞空心化有关,存在潜在隐患。
目前研究最热,发展最迅速的长效蛋白药物技术为Fc融合蛋白技术,Fc片段是IgG保持体内较长半衰期的主要因素,同时可以稳定蛋白,现已用于多种融合蛋白的开发。Fc片段通过两种机制发挥功能,第一种是与细胞表面的Fc受体结合,通过吞噬或裂解作用杀伤细胞并通过抗体依赖性细胞毒(ADCC)途径消化病原体。在四种人IgG亚型中IgG1与IgG3能有效的结合细胞表面的Fc受体,而IgG2和IgG4与受体结合的亲和力较低。第二种是与第一补体成分C1的C1q结合,通过补体依赖的细胞毒(CDC)途径裂解病原体。人IgG1和IgG3能有效的结合C1q从而介导补体级联反应,人IgG2与C1q的结合能力较弱,而人IgG4不与C1q结合。Fc融合蛋白主要是为了增加蛋白药物在体内的半衰期和简化生产工艺降低成本,Fc介导的IgG效应和裂解细胞的功能并不是必要的,甚至会产生副作用。Fc融合蛋白技术不仅如PEG化般增大蛋白药物分子质量降低清除率,更加也能通过FcRn结合域,通过pH敏感性结合力以缓释的方式延长半衰期。这种从基因水平将融合伴侣与效应分子连接在一起,不需要额外的化学修饰,纯化、制备过程简单,质量控制也相对容易,综合来看是非常有前景的一种长效化手段。
发明内容
本发明的首要目的在于提供一种重组人生长激素Fc融合蛋白,编码该融合蛋白的核苷酸的载体、宿主细胞及其用途。由本发明涉及的融合蛋白,作为药物用于治疗儿童生长迟缓和成人的生长激素不足以及重度烧伤等方面,能延长血清半衰期,降低治疗时间内所需的注射给药频率,提高患者的依从性和医患双方的便利性。
本发明提供了一种长效重组人生长激素Fc融合蛋白,所述融合蛋白优选由N端至C端依次为:人生长激素、连接肽、Fc片段(简称rGH-Fc),次优选为Fc片段、连接肽、人生长激素(简称Fc-rGH)。
其中生长激素的氨基酸序列同GenBank:M13438.1中的氨基酸序列,共191个氨基酸。
进一步地,优选的柔性肽接头氨基酸组成的结构通式为(GGGGS)nA,其中n是大于或等于2的整数,在一些实施例,n=3,在另一些实施例中,n=2或4。
n在保护范围内的取值并不影响具体实施例的构建,进行和试验结果。
在一些实施例中,所述人IgG的Fc变体(残基的编号是依据Kabat Eu Index编号)选自下组:
1)至少含有以下两个突变的IgG1 Fc:Leu234Ala,Leu235Ala,Asn297Ala,Val308Pro以及删除C末端Lys,所述IgG1 Fc的核苷酸序列如SEQ ID NO:3所示,氨基酸序列如SEQ ID NO:4所示;
2)至少含有以下两个突变的IgG2 Fc:Asn297Ala,Val308Pro,Pro331Ser以及删除C末端Lys,所述IgG2 Fc的核苷酸序列如SEQ ID NO:5所示,氨基酸序列如SEQ ID NO:6所示;
3)至少含有以下两个突变的IgG4 Fc:至少含有Ser228Pro,Glu233Pro,Leu234Ala或者Leu234Val,Leu235Ala,Asn297Ala,Val308Pro和Arg409Lys以及删除C末端Lys的人IgG4绞链区、CH2和CH3区域,所述IgG4 Fc的核苷酸序列如SEQ ID NO:7所示,氨基酸序列如SEQ ID NO:8所示;
为实现上述目的,本发明采用的技术方案是:
首先确定重组人生长激素Fc融合蛋白的氨基酸序列,在两端加入合适的酶切位点、Koaza序列和引导分泌的信号肽,参考了如中国仓鼠蛋白翻译各密码子利用率的网站或数据库,也参考了一些文献资料在不改变氨基酸序列前提下进行基因编码区密码子优化后,采用大鼠种属偏好密码子,同时去除可能影响抗体表达的序列(如TATA box,AATAATranscription termination site,Cryptic splice sites等),最终反向转换为对应的基因序列,送至基因合成。
将编码融合蛋白的DNA构建入pCDNA3.1载体(来自Invitrogen)中,瞬时转染入Expi293真核细胞,转染5天后收取细胞培养上清液,经Protein A纯化后得到目的融合蛋白。分析目的融合蛋白的纯度,等电点、稳定性、大鼠体内的血浆半衰期、对大鼠血清IGF-1的反应以及其与生长激素受体(GHR)结合后对STAT5信号通路的影响。
在确认目的融合蛋白能刺激大鼠IGF-1反应,在大鼠体内的血浆半衰期明显延长且与生长激素受体(GHR)结合后能有效活化STAT5信号通路后,将编码该融合蛋白的DNA构建入D2M载体,转染CHO-S细胞,利用MTX加压筛选得到高表达rGH-Fc/Fc-rGH的稳定细胞株。
上述技术方案产生的有益效果在于:本发明提供的重组人生长激素融合蛋白,具有特异性结合生长激素受体(GHR),激活下游STAT5信号通路,维持细胞的增殖和分化,且半衰期更长的特点,用于治疗儿童生长迟缓和成人的生长激素不足以及重度烧伤等方面。
附图说明
图1为SDS-PAGE(聚丙烯酰胺凝胶电泳)检测proteinA一步亲和层析纯化后的rGH-Fc/Fc-rGH蛋白,其中左起第一条泳道为Fc-rGH蛋白,第二条泳道为rGH-Fc蛋白,第三条泳道为蛋白Ladder;
图2为SEC检测rGH-Fc蛋白的纯度;
图3为SEC检测Fc-rGH蛋白的纯度;
图4为ELISA检测rGH-Fc/Fc-rGH蛋白与生长激素受体GHR的结合;
图5为ELISA检测rGH-Fc/Fc-rGH蛋白与补体C1q的结合;
图6为荧光素酶报告基因法检测rGH-Fc的生物学活性;
图7为静脉注射等摩尔量rGH-Fc和GH后大鼠血清中IGF-1水平的变化;
图8为静脉注射等摩尔量rGH-Fc和GH后大鼠血清中GH含量的变化;
图9为rGH-Fc工程细胞株的生长曲线,纵坐标分别为细胞数(LCC)和细胞活率(VIA)。
具体实施方式
下面结合实施例对本发明作详细的说明。
实施例1:rGH-Fc/Fc-rGH的表达载体的构建
1.1合成GH基因
NCBI数据库检索GH的氨基酸序列(GenBank Accession:M13438.1),分析其核苷酸序列,针对核苷酸序列进行去相关酶切位点或减少茎环结构等改造,委托全基因合成rGH-Fc全长基因,其核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
1.2PCR扩增XbaI-rGH-Fc-HindIII和XbaI-Fc-rGH-XhoI
设计如下引物:
GH-Fc扩增:以合成的含有GH基因的质粒(pUC57-GH)为模板,以P1和P2为引物,使用Prime STAR HS聚合酶,PCR扩增得到XbaI-GH片段。以已有的IgG-Fc基因的质粒为模板,以P3和P4为引物,使用Prime STARHS聚合酶,PCR扩增得到Fc-HindIII片段。再以上述PCR扩增得到的XbaI-GH和Fc-HindIII为模板,P1和P4为引物,经SOE-PCR扩增得到XbaI-GH-Fc-HindIII。
Fc-GH扩增:以已有的IgG-Fc基因的质粒为模板,以P6和P7为引物,使用PrimeSTARHS聚合酶,PCR扩增得到Fc-pre片段,再以Fc-pre为模板,以P5和P8为引物,扩增得到XbaI-Fc。以合成的含有GH基因的质粒(pUC57-GH)为模板,以P9和P10为引物,使用Prime STAR HS聚合酶,PCR扩增得到GH-XhoI片段。再以上述PCR扩增得到的XbaI-Fc和GH-XhoI为模板,P5和P10为引物,经SOE-PCR扩增得到XbaI-Fc-GH-XhoI。
其扩增体系具体如下:
PCR反应程序,具体如下:
1.3双酶切
XbaI/HindIII酶切载体pCD-cFc(改造自pCDNA3.1+)与目的片段XbaI-GH-Fc-HindIII,XbaI/XhoI双酶切目的片段XbaI-Fc-GH-XhoI和pCD-cFc载体,载体和酶切产物经DNA胶回收后用于连接转化,其中酶切体系如下:
1.4连接转化与测序
取3μl酶连产物,转化入DH-5a感受态细胞,涂布氨苄板后挑克隆送测序,即获得真核表达质粒pCD-rGH-Fc和pCD-Fc-rGH,测序鉴定载体序列正确。
实施例2:rGH-Fc/Fc-rGH蛋白表达与纯化
2.1 rGH-Fc/Fc-rGH的表达与鉴定
向24孔培养板中接种Expi293细胞,接种密度2.5~3×106cells/ml,接种量1ml/孔,转染时细胞活率大于90%。将1μgDNA(pCD-rGH-Fc/Fc-rGH)稀释于50μl OptiMEM,轻轻混匀,再将2.7μl稀释于50μl OptiMEM中,轻轻浑匀,室温孵育5分钟后将所得DNA稀释液和所得ExpiFectamine稀释液混合,轻轻浑匀,室温孵育20分钟,将所得DNA--ExpiFectamine复合物(总体积约100μl)加入培养孔板中,前后摇动培养板使其分布均匀。将细胞放入培养箱继续培养16h,按照5μl/ml的添加比例加入转染Enhancer1,50μl/ml的添加比例加入转染Enhancer2,1μl/ml的添加比例加入抗生素,轻轻混匀,继续培养4天,细胞活率低于50%时,收集全部细胞离心。经ELISA鉴定确认rGH-Fc/Fc-rGH融合蛋白表达后,扩大转染体系至30ml(操作同上),转染5天后收取上清液纯化蛋白用。
2.2rGH-Fc/Fc-rGH蛋白的纯化与纯度鉴定
收集培养基上清液,用proteinA树脂纯化rGH-Fc/Fc-rGH,随后用截留分子量为30kDa的超滤离心管超滤浓缩rGH-Fc/Fc-rGH,经SDS-PAGE鉴定结果如附图1所示,其中泳道1为Fc-rGH,泳道2为rGH-Fc,泳道3为蛋白Ladder,表明rGH-Fc/Fc-rGH的分子量大小与理论(97KD)一致且纯度较高。附图2和图3分别为rGH-Fc和Fc-rGH蛋白的SEC鉴定图,积分结果显示主峰的纯度均大于95%。
实施例3:rGH-Fc/Fc-rGH蛋白与GHR的结合
用PBS稀释生长激素受体蛋白(GHR)至1μg/ml,4℃铺板过夜。TPBS洗涤3次,37℃烘箱封闭1h,TPBS洗3次后加入梯度稀释的rGH-Fc/Fc-rGH融合蛋白(50,25,12.5,6.25,3.12,1.56,0.78,0.39,0.19nM),每孔100μl,室温震荡孵育1小时。TPBS洗涤3次后加入绵羊抗人Fc-HRP(1:4000稀释),每孔100μl,室温震荡孵育1小时,TPBS洗3次,加入OPD显色,室温反应10min后加入1MH2SO4以终止反应,每孔100μl,在490nm处测量吸光度。检测结果如图4所示,结果表明rGH-Fc和Fc-rGH均能与GHR结合,且呈现浓度梯度依赖趋势。
实施例4:rGH-Fc/Fc-rGH蛋白与C1q的结合
用PBS稀释rGH-Fc/Fc-rGH蛋白和Herceptin抗体抗原稀释至8,4,2.000,1,0.5,0.25,0.125,0μg/mL,加入96孔板中,每孔100μl,4℃铺板过夜。TPBS洗涤3次,37℃烘箱封闭1h,TPBS洗3次后加入2μg/mL人C1q蛋白,每孔50μl,室温震荡孵育1小时。TPBS洗涤3次后加入绵羊抗人C1q-HRP(1:500稀释),每孔100μl,室温震荡孵育1小时,TPBS洗3次,加入OPD显色,室温反应10min后加入1MH2SO4以终止反应,每孔100μl,在490nm处测量吸光度。检测结果如图5所示,结果表明IgG1亚型的Herceptin与C1q蛋白的结合呈浓度梯度依赖趋势,而rGH-Fc/Fc-rGH蛋白不与C1q结合。
实施例5:rGH-Fc蛋白的生物学活性测定
在HEK293细胞中转入含有STAT5启动子的荧光素酶报告基因和GHR受体质粒,获得HEK293-GHR-Luc细胞株。取对数生长期的HEK293-GHR-Luc细胞,胰酶消化后用DMEM+10%FBS+1%PS+2μg/ml of Puromycin培养基重悬细胞使细胞浓度达到6ⅹ105cells/ml。在96孔板中加入50μl细胞混悬液,即30000cells/well,并在96孔板四周加入100μl PBS缓冲液,CO2培养箱培养16-24h。用不含酚红的DMEM+10%FBS培养基稀释rGH-Fc蛋白至200μg/ml,再做8倍梯度稀释,共8个梯度,取50μl的稀释的蛋白加入96孔板中(即使其终浓度为100,14.2857,2.0408,0.2915,0.0416,0.0059,0.0008,0.0001μg/ml),随后放入CO2培养箱中培养5h后每孔加入100μl已经达到室温的Promega Bio-GloTM luciferase Assary System,并放置在室温用振荡器震荡10min后上机检测。测试结果如附图5所示。附图5表明:HEK293-GHR-Luc经GH-Fc刺激后,GH-Fc与受体GHR结合后激活JAK2-STATs信号通路,从而激活STAT5反应元件下游的荧光素酶基因的转录,荧光素酶基因表达量的强弱与结合至靶细胞膜上GHR的GH-Fc含量呈正相关。
实施例6:rGH-Fc蛋白与对大鼠IGF-1水平的影响
将雄性成熟Sprague Dawley大鼠(约7周龄,体重130-180g)分成3组,每组5只,分别静脉注射等体积的生理盐水、15nmol生长激素水针和15nmol重组人生长激素蛋白(rGH-Fc),单次给药给药0,1,4,12,24,48,96h后取血样,用Rat IGF-1 ELISAKit(厂商:Elabscience,货号:E-EL-R0010c)检测大鼠血清中的IGF-1含量。具体操作如下:首先取出包被有抗IGF-1抗体的酶标板上平衡至室温,加入100μL梯度稀释的rat IGF-1标准品蛋白(100,50,25,12.5,6.25,3.13,1.56,0ng/mL)和待测样品,37℃孵育90分钟后弃去液体,甩干后加入生物素化抗体工作液100μL,37℃温育1小时。洗板3次后每孔加酶结合物工作液100μL,37℃温育30分钟。洗板5次再每孔加90μL底物溶液TMB,37℃避光孵育15分钟左右每孔加终止液50μL,立即用酶标仪在450nm波长测量各孔的光密度(OD值),根据标准GH蛋白的吸光值计算样品的IGF-1浓度。检测结果如附图6所示,结果表明,静脉注射GH和rGH-Fc后均能引起大鼠血清中IGF-1水平的短暂上升。
实施例7:rGH-Fc蛋白在大鼠体内的血浆半衰期
将雄性成熟Sprague Dawley大鼠(约7周龄,体重130-180g)分成3组,每组5只,分别静脉注射等体积的生理盐水、15nmol生长激素水针和15nmol重组人生长激素蛋白(rGH-Fc),单次给药给药0、0.25、1、2、4、8、12、24、32、48、72、96、144、192h后取血样,用ELISA检测大鼠血清中的GH含量,具体操作如下:首先用PBS稀释抗GH抗体4D9(1:1000),加入96孔板中,每孔100μl,4℃铺板过夜。TPBS洗涤3次,37℃烘箱封闭1h,TPBS洗3次后加入梯度稀释的标准GH蛋白和待测样品(2000,1000,500,250,125,62.5,31.25,15.625ng/mL),每孔100μl,室温震荡孵育1小时。TPBS洗涤3次后加入6A4-HRP(1:1000),每孔100μl,室温震荡孵育1小时,TPBS洗3次,加入OPD显色,室温反应10min后加入1MH2SO4以终止反应,每孔100μl,在490nm处测量吸光度,根据标准GH蛋白的吸光值计算样品的GH和rGH-Fc浓度。检测结果如附图7所示,结果表明,rGH-Fc在大鼠体内的额血浆半衰期较GH更长。
实施例8:高表达rGH-Fc蛋白的工程细胞株的构建与筛选
将核苷酸优化后的rGH-Fc构建入DN2载体,经PvuI酶切线性化后转染入CHO细胞,用50~100~200~500~1000nM MTX加压后以0.5cell/well铺板,使用ELISA法检测培养上清液中融合蛋白的含量进行筛选,得到表达量相对较高且细胞生长状态较好的5个优选一级亚克隆,将一级亚克隆以以0.3cell/wel铺板筛选二级亚克隆,筛选后最终得到1个优选克隆和2个次优克隆。优选克隆培养14天的细胞生长曲线如图7所示,上清液中rGH-Fc的含量>2g/L。
本文中应用了具体个例对本发明的原理及实施方式进行了阐述,以上实例的说明只是用于帮助理解本发明的方法及其核心思想。以上所述仅是本发明的优选实施方式,应当指出,由于文字表达的有限性,而客观上存在无限的具体结构,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进、润饰或变化,也可以将上述技术特征以适当的方式进行组合;这些改进润饰、变化或组合,或未经改进将发明的构思和技术方案直接应用于其它场合的,均应视为本发明的保护范围。
SEQUENCE LISTING
<110> 安徽安科生物工程(集团)股份有限公司
<120> 重组人生长激素Fc融合蛋白研制和用途及其工程细胞株
<130> 2020
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 651
<212> DNA
<213> Artificial Sequence
<220>
<223> 生长激素核苷酸序列
<400> 1
atggccaccg gcagccggac ctctctgctc ctggccttcg gactgctctg cctgccttgg 60
ctgcaggaag gcagcgcttt ccctacaatc cctctgagcc ggctgttcga taacgccatg 120
ctgagagccc acaggctgca ccagctggcc ttcgacacct accaggagtt cgaggaagcc 180
tacatcccta aggagcagaa gtacagcttc ctgcagaacc ctcagaccag cctgtgtttc 240
tctgagtcca tccctacccc aagcaaccgg gaggaaaccc agcagaagag caacctggag 300
ctgctcagaa tctccctgct cctgatccag agctggctgg agcctgtgca gttcctgcgg 360
agcgtgttcg ccaacagcct ggtgtacgga gccagcgaca gtaacgtgta cgacctgctc 420
aaggacctgg aggaaggcat ccagaccctg atgggcagac tggaggacgg cagccctcgg 480
acaggccaga tcttcaagca gacctacagc aagttcgaca caaacagcca caacgatgac 540
gccctgctca agaactacgg cctgctctac tgcttcagaa aggacatgga taaggtggaa 600
acattcctga gaatcgtgca gtgcaggtcc gtggaaggca gctgcggctt c 651
<210> 2
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> 生长激素氨基酸序列
<400> 2
Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu
1 5 10 15
Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu
20 25 30
Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln
35 40 45
Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys
50 55 60
Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe
65 70 75 80
Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys
85 90 95
Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp
100 105 110
Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val
115 120 125
Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu
130 135 140
Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg
145 150 155 160
Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser
165 170 175
His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe
180 185 190
Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys
195 200 205
Arg Ser Val Glu Gly Ser Cys Gly Phe
210 215
<210> 3
<211> 699
<212> DNA
<213> Artificial Sequence
<220>
<223> IgG1 Fc核苷酸序列
<400> 3
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt ctcctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaaatga 699
<210> 4
<211> 232
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG1 Fc氨基酸序列
<400> 4
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Ser
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 5
<211> 687
<212> DNA
<213> Artificial Sequence
<220>
<223> IgG2 Fc核苷酸序列
<400> 5
gagaggaaat gctgtgtgga gtgccctccc tgtcctgctc ctcccgtggc tggacctagc 60
gtgttcctgt tccctcccaa gcctaaagac accctgatga tcagccggac ccctgaagtg 120
acatgcgtgg tcgtggacgt gtcccacgaa gaccctgagg tgcagttcaa ctggtacgtg 180
gatggcgtgg aagtgcacaa cgccaagaca aaacctcggg aggaacagtt caacagcacc 240
ttcagagtgg tgtccgtgct gaccgtggtc caccaggact ggttgaacgg caaggagtac 300
aaatgcaagg tgagcaacaa gggcctccct gctcccatcg agaagaccat ctccaagacc 360
aagggccagc ctcgggaacc tcaggtgtac actctgcctc caagcaggga ggaaatgacc 420
aagaatcagg tgtctctgac ctgcctcgtg aagggcttct accctagcga catttccgtg 480
gagtgggaat ctaacggaca gcctgagaac aattacaaga ccacacctcc catgcttgac 540
tccgacggca gcttctttct gtactctaag ctcaccgtgg acaaaagcag gtggcagcag 600
ggcaatgtgt tctcttgtag cgtcatgcac gaggccctgc acaaccatta cacccagaag 660
agcctgtccc tctctcctgg caagtga 687
<210> 6
<211> 228
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG2 Fc氨基酸序列
<400> 6
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
65 70 75 80
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro
100 105 110
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Pro Gly Lys
225
<210> 7
<211> 690
<212> DNA
<213> Artificial Sequence
<220>
<223> IgG4 Fc核苷酸序列
<400> 7
gaatctaagt acggccctcc ctgccccagc tgtcctgccc ctgaatttct gggcggaccc 60
tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120
gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240
acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300
tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360
gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420
accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480
gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540
gactccgacg gctccttctt tctgtattct cgcctgaccg tggacaagtc ccggtggcag 600
gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660
aagtccctgt ccctgtctct gggaaagtga 690
<210> 8
<211> 229
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG4 Fc氨基酸序列
<400> 8
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 9
<211> 1917
<212> DNA
<213> Artificial Sequence
<220>
<223> 生长激素受体核苷酸序列
<400> 9
atggatctct ggcagctgct gttgaccttg gcactggcag gatcaagtga tgctttttct 60
ggaagtgagg ccacagcagc tatccttagc agagcaccct ggagtctgca aagtgttaat 120
ccaggcctaa agacaaattc ttctaaggag cctaaattca ccaagtgccg ttcacctgag 180
cgagagactt tttcatgcca ctggacagat gaggttcatc atggtacaaa gaacctagga 240
cccatacagc tgttctatac cagaaggaac actcaagaat ggactcaaga atggaaagaa 300
tgccctgatt atgtttctgc tggggaaaac agctgttact ttaattcatc gtttacctcc 360
atctggatac cttattgtat caagctaact agcaatggtg gtacagtgga tgaaaagtgt 420
ttctctgttg atgaaatagt gcaaccagat ccacccattg ccctcaactg gactttactg 480
aacgtcagtt taactgggat tcatgcagat atccaagtga gatgggaagc accacgcaat 540
gcagatattc agaaaggatg gatggttctg gagtatgaac ttcaatacaa agaagtaaat 600
gaaactaaat ggaaaatgat ggaccctata ttgacaacat cagttccagt gtactcattg 660
aaagtggata aggaatatga agtgcgtgtg agatccaaac aacgaaactc tggaaattat 720
ggcgagttca gtgaggtgct ctatgtaaca cttcctcaga tgagccaatt tacatgtgaa 780
gaagatttct actttccatg gctcttaatt attatctttg gaatatttgg gctaacagtg 840
atgctatttg tattcttatt ttctaaacag caaaggatta aaatgctgat tctgccccca 900
gttccagttc caaagattaa aggaatcgat ccagatctcc tcaaggaagg aaaattagag 960
gaggtgaaca caatcttagc cattcatgat agctataaac ccgaattcca cagtgatgac 1020
tcttgggttg aatttattga gctagatatt gatgagccag atgaaaagac tgaggaatca 1080
gacacagaca gacttctaag cagtgaccat gagaaatcac atagtaacct aggggtgaag 1140
gatggcgact ctggacgtac cagctgttgt gaacctgaca ttctggagac tgatttcaat 1200
gccaatgaca tacatgaggg tacctcagag gttgctcagc cacagaggtt aaaaggggaa 1260
gcagatctct tatgccttga ccagaagaat caaaataact caccttatca tgatgcttgc 1320
cctgctactc agcagcccag tgttatccaa gcagagaaaa acaaaccaca accacttcct 1380
actgaaggag ctgagtcaac tcaccaagct gcccatattc agctaagcaa tccaagttca 1440
ctgtcaaaca tcgactttta tgcccaggtg agcgacatta caccagcagg tagtgtggtc 1500
ctttccccgg gccaaaagaa taaggcaggg atgtcccaat gtgacatgca cccggaaatg 1560
gtctcactct gccaagaaaa cttccttatg gacaatgcct acttctgtga ggcagatgcc 1620
aaaaagtgca tccctgtggc tcctcacatc aaggttgaat cacacataca gccaagctta 1680
aaccaagagg acatttacat caccacagaa agccttacca ctgctgctgg gaggcctggg 1740
acaggagaac atgttccagg ttctgagatg cctgtcccag actatacctc cattcatata 1800
gtacagtccc cacagggcct catactcaat gcgactgcct tgcccttgcc tgacaaagag 1860
tttctctcat catgtggcta tgtgagcaca gaccaactga acaaaatcat gccttag 1917
<210> 10
<211> 638
<212> PRT
<213> Artificial Sequence
<220>
<223> 生长激素受体氨基酸序列
<400> 10
Met Asp Leu Trp Gln Leu Leu Leu Thr Leu Ala Leu Ala Gly Ser Ser
1 5 10 15
Asp Ala Phe Ser Gly Ser Glu Ala Thr Ala Ala Ile Leu Ser Arg Ala
20 25 30
Pro Trp Ser Leu Gln Ser Val Asn Pro Gly Leu Lys Thr Asn Ser Ser
35 40 45
Lys Glu Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe
50 55 60
Ser Cys His Trp Thr Asp Glu Val His His Gly Thr Lys Asn Leu Gly
65 70 75 80
Pro Ile Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln
85 90 95
Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys
100 105 110
Tyr Phe Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys
115 120 125
Leu Thr Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val Asp
130 135 140
Glu Ile Val Gln Pro Asp Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu
145 150 155 160
Asn Val Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu
165 170 175
Ala Pro Arg Asn Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr
180 185 190
Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp
195 200 205
Pro Ile Leu Thr Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys
210 215 220
Glu Tyr Glu Val Arg Val Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr
225 230 235 240
Gly Glu Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Gln
245 250 255
Phe Thr Cys Glu Glu Asp Phe Tyr Phe Pro Trp Leu Leu Ile Ile Ile
260 265 270
Phe Gly Ile Phe Gly Leu Thr Val Met Leu Phe Val Phe Leu Phe Ser
275 280 285
Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro
290 295 300
Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu
305 310 315 320
Glu Val Asn Thr Ile Leu Ala Ile His Asp Ser Tyr Lys Pro Glu Phe
325 330 335
His Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Glu
340 345 350
Pro Asp Glu Lys Thr Glu Glu Ser Asp Thr Asp Arg Leu Leu Ser Ser
355 360 365
Asp His Glu Lys Ser His Ser Asn Leu Gly Val Lys Asp Gly Asp Ser
370 375 380
Gly Arg Thr Ser Cys Cys Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn
385 390 395 400
Ala Asn Asp Ile His Glu Gly Thr Ser Glu Val Ala Gln Pro Gln Arg
405 410 415
Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn
420 425 430
Asn Ser Pro Tyr His Asp Ala Cys Pro Ala Thr Gln Gln Pro Ser Val
435 440 445
Ile Gln Ala Glu Lys Asn Lys Pro Gln Pro Leu Pro Thr Glu Gly Ala
450 455 460
Glu Ser Thr His Gln Ala Ala His Ile Gln Leu Ser Asn Pro Ser Ser
465 470 475 480
Leu Ser Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala
485 490 495
Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Met Ser
500 505 510
Gln Cys Asp Met His Pro Glu Met Val Ser Leu Cys Gln Glu Asn Phe
515 520 525
Leu Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile
530 535 540
Pro Val Ala Pro His Ile Lys Val Glu Ser His Ile Gln Pro Ser Leu
545 550 555 560
Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Ala Ala
565 570 575
Gly Arg Pro Gly Thr Gly Glu His Val Pro Gly Ser Glu Met Pro Val
580 585 590
Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Ile
595 600 605
Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser
610 615 620
Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro
625 630 635
<110> 安徽安科生物工程(集团)股份有限公司
<120> 重组人生长激素Fc融合蛋白研制和用途及其工程细胞株
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 651
<212> DNA
<213> Artificial Sequence
<220>
<223> 生长激素核苷酸序列
<400> 1
atggccaccg gcagccggac ctctctgctc ctggccttcg gactgctctg cctgccttgg 60
ctgcaggaag gcagcgcttt ccctacaatc cctctgagcc ggctgttcga taacgccatg 120
ctgagagccc acaggctgca ccagctggcc ttcgacacct accaggagtt cgaggaagcc 180
tacatcccta aggagcagaa gtacagcttc ctgcagaacc ctcagaccag cctgtgtttc 240
tctgagtcca tccctacccc aagcaaccgg gaggaaaccc agcagaagag caacctggag 300
ctgctcagaa tctccctgct cctgatccag agctggctgg agcctgtgca gttcctgcgg 360
agcgtgttcg ccaacagcct ggtgtacgga gccagcgaca gtaacgtgta cgacctgctc 420
aaggacctgg aggaaggcat ccagaccctg atgggcagac tggaggacgg cagccctcgg 480
acaggccaga tcttcaagca gacctacagc aagttcgaca caaacagcca caacgatgac 540
gccctgctca agaactacgg cctgctctac tgcttcagaa aggacatgga taaggtggaa 600
acattcctga gaatcgtgca gtgcaggtcc gtggaaggca gctgcggctt c 651
<210> 2
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> 生长激素氨基酸序列
<400> 2
Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu
1 5 10 15
Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu
20 25 30
Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln
35 40 45
Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys
50 55 60
Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe
65 70 75 80
Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys
85 90 95
Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp
100 105 110
Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val
115 120 125
Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu
130 135 140
Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg
145 150 155 160
Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser
165 170 175
His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe
180 185 190
Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys
195 200 205
Arg Ser Val Glu Gly Ser Cys Gly Phe
210 215
<210> 3
<211> 687
<212> DNA
<213> Artificial Sequence
<220>
<223> IgG4 Fc变体1核苷酸序列
<400> 3
gaatctaagt acggccctcc ctgcccccct tgtcctgccc ctgaagccgc tggcggaccc 60
tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120
gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240
acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300
tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360
gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420
accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480
gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540
gactccgacg gctccttctt tctgtattct aagctgaccg tggacaagtc ccggtggcag 600
gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660
aagtccctgt ccctgtctct gggatag 687
<210> 4
<211> 228
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG4 Fc变体1氨基酸序列
<400> 4
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly
225
<210> 5
<211> 687
<212> DNA
<213> Artificial Sequence
<220>
<223> IgG4 Fc变体2核苷酸序列
<400> 5
gaatctaagt acggccctcc ctgcccccct tgtcctgccc ctcctgtggc tggcggaccc 60
tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120
gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240
acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300
tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360
gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420
accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480
gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540
gactccgacg gctccttctt tctgtattct cgcctgaccg tggacaagtc ccggtggcag 600
gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660
aagtccctgt ccctgtctct gggatag 687
<210> 6
<211> 228
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG4 Fc变体2氨基酸序列
<400> 6
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly
225
<210> 7
<211> 1917
<212> DNA
<213> Artificial Sequence
<220>
<223> 生长激素受体核苷酸序列
<400> 7
atggatctct ggcagctgct gttgaccttg gcactggcag gatcaagtga tgctttttct 60
ggaagtgagg ccacagcagc tatccttagc agagcaccct ggagtctgca aagtgttaat 120
ccaggcctaa agacaaattc ttctaaggag cctaaattca ccaagtgccg ttcacctgag 180
cgagagactt tttcatgcca ctggacagat gaggttcatc atggtacaaa gaacctagga 240
cccatacagc tgttctatac cagaaggaac actcaagaat ggactcaaga atggaaagaa 300
tgccctgatt atgtttctgc tggggaaaac agctgttact ttaattcatc gtttacctcc 360
atctggatac cttattgtat caagctaact agcaatggtg gtacagtgga tgaaaagtgt 420
ttctctgttg atgaaatagt gcaaccagat ccacccattg ccctcaactg gactttactg 480
aacgtcagtt taactgggat tcatgcagat atccaagtga gatgggaagc accacgcaat 540
gcagatattc agaaaggatg gatggttctg gagtatgaac ttcaatacaa agaagtaaat 600
gaaactaaat ggaaaatgat ggaccctata ttgacaacat cagttccagt gtactcattg 660
aaagtggata aggaatatga agtgcgtgtg agatccaaac aacgaaactc tggaaattat 720
ggcgagttca gtgaggtgct ctatgtaaca cttcctcaga tgagccaatt tacatgtgaa 780
gaagatttct actttccatg gctcttaatt attatctttg gaatatttgg gctaacagtg 840
atgctatttg tattcttatt ttctaaacag caaaggatta aaatgctgat tctgccccca 900
gttccagttc caaagattaa aggaatcgat ccagatctcc tcaaggaagg aaaattagag 960
gaggtgaaca caatcttagc cattcatgat agctataaac ccgaattcca cagtgatgac 1020
tcttgggttg aatttattga gctagatatt gatgagccag atgaaaagac tgaggaatca 1080
gacacagaca gacttctaag cagtgaccat gagaaatcac atagtaacct aggggtgaag 1140
gatggcgact ctggacgtac cagctgttgt gaacctgaca ttctggagac tgatttcaat 1200
gccaatgaca tacatgaggg tacctcagag gttgctcagc cacagaggtt aaaaggggaa 1260
gcagatctct tatgccttga ccagaagaat caaaataact caccttatca tgatgcttgc 1320
cctgctactc agcagcccag tgttatccaa gcagagaaaa acaaaccaca accacttcct 1380
actgaaggag ctgagtcaac tcaccaagct gcccatattc agctaagcaa tccaagttca 1440
ctgtcaaaca tcgactttta tgcccaggtg agcgacatta caccagcagg tagtgtggtc 1500
ctttccccgg gccaaaagaa taaggcaggg atgtcccaat gtgacatgca cccggaaatg 1560
gtctcactct gccaagaaaa cttccttatg gacaatgcct acttctgtga ggcagatgcc 1620
aaaaagtgca tccctgtggc tcctcacatc aaggttgaat cacacataca gccaagctta 1680
aaccaagagg acatttacat caccacagaa agccttacca ctgctgctgg gaggcctggg 1740
acaggagaac atgttccagg ttctgagatg cctgtcccag actatacctc cattcatata 1800
gtacagtccc cacagggcct catactcaat gcgactgcct tgcccttgcc tgacaaagag 1860
tttctctcat catgtggcta tgtgagcaca gaccaactga acaaaatcat gccttag 1917
<210> 8
<211> 638
<212> PRT
<213> Artificial Sequence
<220>
<223> 生长激素受体氨基酸序列
<400> 8
Met Asp Leu Trp Gln Leu Leu Leu Thr Leu Ala Leu Ala Gly Ser Ser
1 5 10 15
Asp Ala Phe Ser Gly Ser Glu Ala Thr Ala Ala Ile Leu Ser Arg Ala
20 25 30
Pro Trp Ser Leu Gln Ser Val Asn Pro Gly Leu Lys Thr Asn Ser Ser
35 40 45
Lys Glu Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe
50 55 60
Ser Cys His Trp Thr Asp Glu Val His His Gly Thr Lys Asn Leu Gly
65 70 75 80
Pro Ile Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln
85 90 95
Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys
100 105 110
Tyr Phe Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys
115 120 125
Leu Thr Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val Asp
130 135 140
Glu Ile Val Gln Pro Asp Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu
145 150 155 160
Asn Val Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu
165 170 175
Ala Pro Arg Asn Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr
180 185 190
Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp
195 200 205
Pro Ile Leu Thr Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys
210 215 220
Glu Tyr Glu Val Arg Val Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr
225 230 235 240
Gly Glu Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Gln
245 250 255
Phe Thr Cys Glu Glu Asp Phe Tyr Phe Pro Trp Leu Leu Ile Ile Ile
260 265 270
Phe Gly Ile Phe Gly Leu Thr Val Met Leu Phe Val Phe Leu Phe Ser
275 280 285
Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro
290 295 300
Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu
305 310 315 320
Glu Val Asn Thr Ile Leu Ala Ile His Asp Ser Tyr Lys Pro Glu Phe
325 330 335
His Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Glu
340 345 350
Pro Asp Glu Lys Thr Glu Glu Ser Asp Thr Asp Arg Leu Leu Ser Ser
355 360 365
Asp His Glu Lys Ser His Ser Asn Leu Gly Val Lys Asp Gly Asp Ser
370 375 380
Gly Arg Thr Ser Cys Cys Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn
385 390 395 400
Ala Asn Asp Ile His Glu Gly Thr Ser Glu Val Ala Gln Pro Gln Arg
405 410 415
Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn
420 425 430
Asn Ser Pro Tyr His Asp Ala Cys Pro Ala Thr Gln Gln Pro Ser Val
435 440 445
Ile Gln Ala Glu Lys Asn Lys Pro Gln Pro Leu Pro Thr Glu Gly Ala
450 455 460
Glu Ser Thr His Gln Ala Ala His Ile Gln Leu Ser Asn Pro Ser Ser
465 470 475 480
Leu Ser Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala
485 490 495
Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Met Ser
500 505 510
Gln Cys Asp Met His Pro Glu Met Val Ser Leu Cys Gln Glu Asn Phe
515 520 525
Leu Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile
530 535 540
Pro Val Ala Pro His Ile Lys Val Glu Ser His Ile Gln Pro Ser Leu
545 550 555 560
Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Ala Ala
565 570 575
Gly Arg Pro Gly Thr Gly Glu His Val Pro Gly Ser Glu Met Pro Val
580 585 590
Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Ile
595 600 605
Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser
610 615 620
Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro
Claims (15)
1.一种重组人生长激素Fc融合蛋白,其特征在于,所述融合蛋白由人生长激素、连接肽和Fc片段组成,所述融合蛋白由N端至C端依次为:人生长激素、连接肽、Fc片段,或者所述融合蛋白由N端至C端依次为Fc片段、连接肽、人生长激素。
2.一种根据权利要求1所述的重组人生长激素Fc融合蛋白,其特征在于,所述编码人生长激素基因的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
3.一种根据权利要求1所述的重组人生长激素Fc融合蛋白,其特征在于,所述连接肽为柔性连接肽,所述柔性肽接头氨基酸组成的结构通式为(GGGGS)nA,其中n是大于或等于2的整数。
4.一种根据权利要求1所述的重组人生长激素Fc融合蛋白,其特征在于,所述Fc片段为人IgG Fc变体。
5.一种根据权利要求4所述的重组人生长激素Fc融合蛋白,其特征在于,所述的人IgGFc变体选自以下一组或多组:
1)至少含有以下两个突变的IgG1 Fc:Leu234Ala,Leu235Ala,Asn297Ala,Val308Pro以及删除C末端Lys,所述IgG1 Fc的核苷酸序列如SEQ ID NO:3所示,氨基酸序列如SEQ IDNO:4所示;
2)至少含有以下两个突变的IgG2 Fc:Asn297Ala,Val308Pro,Pro331Ser以及删除C末端Lys,所述IgG2 Fc的核苷酸序列如SEQ ID NO:5所示,氨基酸序列如SEQ ID NO:6所示;
3)至少含有以下两个突变的IgG4 Fc:至少含有Ser228Pro,Glu233Pro,Leu234Ala或者Leu234Val,Leu235Ala,Asn297Ala,Val308Pro和Arg409Lys以及删除C末端Lys的人IgG4绞链区、CH2和CH3区域,所述IgG4 Fc的核苷酸序列如SEQ ID NO:7所示,氨基酸序列如SEQ IDNO:8所示。
6.一种多核苷酸,其特征在于,编码权利要求1-5任一权利要求所述的重组人生长激素Fc融合蛋白的氨基酸。
7.一种载体或载体组,其特征在于,包含权利要求6所述的多核苷酸。
8.一种宿主细胞,其特征在于,包含权利要求7所述的载体或载体组,所述宿主细胞是原核的或真核的,为酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞中的一种或多种。
9.一种表达权利要求1至5任一项所述融合蛋白的阳性克隆及亚克隆的筛选方法,其特征在于,通过逐渐提高氨甲喋呤(amethopterin,MTX)浓度,从96板→24孔板→6孔板→25cm2培养瓶→125ml摇瓶,采用ELISA法逐步筛选出细胞株,将最终筛选细胞株命名为rhGH-Fc/Fc-rGH细胞株,表达的目的蛋白为rhGH-Fc/Fc-rGH重组蛋白。
10.一种药物组合物,其特征在于,包含权利要求1-5任意一项所述的重组人生长激素Fc融合蛋白,或包含权利要求7所述的载体或载体组,以及药学上可接受的载体。
11.一种试剂盒,其特征在于,所述试剂盒包含如权利要求1-5中任意一项所述的重组人生长激素Fc融合蛋白或如权利要求10所述的药物组合物。
12.一种生产重组人生长激素Fc融合蛋白的方法,其特征在于,所述方法包括在表达所述多核苷酸的条件下培养如权利要求8所述的宿主细胞。
13.一种根据权利要求1-5任一项所述的重组人生长激素Fc融合蛋白的用途,其特征在于,所述融合蛋白诱导延长的IGF-1反应。
14.一种生长激素受体,其特征在于,所述生长激素受体能与权利要求1-5任一项所述的重组人生长激素Fc融合蛋白结合,并激活JAK2-STATs信号通路,所述编码生长激素受体的核苷酸序列如SEQ ID NO:9所示,氨基酸序列如SEQ ID NO:10所示。
15.一种根据权利要求1-5任一项所述的重组人生长激素Fc融合蛋白或根据权利要求8所述的宿主细胞或包含权利要求8所述的宿主细胞的菌株群的用途,其特征在于,所述融合蛋白或所述宿主细胞或所述菌株群用于延长血清半衰期,降低治疗时间内所需的注射给药频率,提高患者的依从性和医患双方的便利性。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113880954A (zh) * | 2021-09-29 | 2022-01-04 | 江苏大学 | 一种重组人生长激素及其构建方法和应用 |
CN114010765A (zh) * | 2021-08-30 | 2022-02-08 | 上海延立药业有限公司 | 一种长效人生长激素 |
CN114426569A (zh) * | 2022-01-25 | 2022-05-03 | 宁波甲贝医药科技有限公司 | 一种用于构建融合蛋白的连接肽 |
CN115400076A (zh) * | 2022-08-17 | 2022-11-29 | 安徽安科生物工程(集团)股份有限公司 | 重组人生长激素-Fc融合蛋白注射液制剂配方 |
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CN115400076A (zh) * | 2022-08-17 | 2022-11-29 | 安徽安科生物工程(集团)股份有限公司 | 重组人生长激素-Fc融合蛋白注射液制剂配方 |
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