CN101698852B - 具有cd137l功能的蛋白或多肽及其基因和应用 - Google Patents
具有cd137l功能的蛋白或多肽及其基因和应用 Download PDFInfo
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Abstract
本发明属于基因工程领域，公开了具有CD137L功能的蛋白或多肽及其基因和应用。该蛋白或多肽为CD137L胞外区蛋白和His tag标签通过0-3个氨基酸连接肽融合而成。它具有SEQ ID NO.17至SEQ ID NO.32所示之一的氨基酸序列。编码该蛋白或多肽的基因相对应地具有SEQ ID NO.1至SEQ ID NO.16所示之一的核苷酸序列。该蛋白或多肽及其基因可用于制备调节机体免疫力、细胞增殖和机体细胞因子的合成与分泌的药物中的应用。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种具有CD137L功能的蛋白或多肽及其基因，以及含有该基因的表达载体以及该载体转化的菌株，还涉及该蛋白或多肽的制备方法，同时涉及具有CD137L功能的蛋白或多肽及其基因在制备调节机体免疫力、细胞增殖和机体细胞因子的合成与分泌的药物中的应用。
背景技术
大量研究明确表明，在人和动物体内对恶性肿瘤存在着某种程度的免疫反应。肿瘤患者免疫系统的细胞能够识别肿瘤细胞表达的抗原，如组织分化抗原、癌胚抗原以及突变基因的产物等等。多项临床研究显示，肿瘤浸润性淋巴细胞有良好的预后意义。此外，免疫调节剂，如细胞因子、微生物产品、癌症疫苗以及过继免疫治疗对很多病人的肿瘤消除有显著作用。虽然有这些反应，但仅仅依靠病人自身免疫系统并不能有效消除肿瘤细胞。导致这种失败的原因大致可分为三类：1)肿瘤抗原的表达变化或MHC I类物质表达减少导致免疫细胞识别肿瘤细胞的能力受损；2)肿瘤细胞抑制性细胞因子的分泌导致肿瘤微环境产生免疫抑制(如TGF-α)；3)由于肿瘤细胞所表达的共刺激分子相对较少，所以无法有效刺激T细胞，从而导致肿瘤免疫原性较差。随着对肿瘤抗原识别及免疫效应功能要求的了解，我们认识到可通过一个辅助分子来提供共刺激信号以提高抗肿瘤免疫反应。由于肿瘤抗原特异性T细胞需要协同刺激来启动和维持效应器的功能，因此，靶向协同刺激分子的治疗可用于调节和增强对肿瘤的免疫反应。
T细胞的激活需要两个信号的参与：一个是TCR接受抗原递呈细胞(APC)传导的MHC-抗原肽信号，另一个是细胞膜表面黏附分子提供的协同刺激信号(或称第二信号)。增强T淋巴细胞活化所需要的协同刺激信号是增强抗肿瘤免疫的重要方法。CD137是继CD28/B7之外新发现的另一重要的T细胞协同刺激分子，CD137与CD137配体(CD137L)在维持T细胞，尤其是T细胞的活化、增殖的方面具有重要的意义。
CD137(亦命名为4-1BB)是近年发现的TNFR家族的新成员，在调节细胞增殖、分化、凋亡中起重要作用。CD137是I型跨膜糖蛋白，由信号肽、胞外区、疏水区及胞内区构成，以单体或二聚体形式表达于活化的T细胞表面。它不仅表达于活化的T淋巴细胞、NK细胞、NKT细胞、CD4/CD25调节T细胞，也可表达于活化的胸腺细胞和上皮内淋巴细胞。此外，CD137还可表达于一些非淋巴细胞，如单核细胞、粒细胞及树突状细胞(DC)等，提示CD137可能在免疫调节中起重要作用。其配体CD137L(亦命名为4-1BBL)首先由Goodwin等利用表达筛选法在小鼠胸腺瘤细胞中分离出，随后又在人CD4+T细胞克隆中分离得到。CD137L也是TNFR家族的成员，是存在于细胞表面的II型膜蛋白，与其他TNF家族有相似的C-端氨基酸，分子量为34KD。其膜型也是一种可诱导的细胞表面抗原，可表达于多种抗原呈递细胞(APC)、各种肿瘤细胞及活化诱导的T细胞上。人和小鼠的CD137L氨基酸序列有36％的同源性。
CD137分子对T细胞活化的重要作用已得到充分认可。在鼠和人T细胞中均可观察到，仅有亚适量CD3抗体(第一信号)的存在下，CD137便可诱导T细胞增值和细胞因子的合成(主要是IFN-α)，以及抑制活化细胞的死亡。协同刺激信号可通过提高抗原特异性和效应CD8+T细胞的数量来增强效应功能(例如，干扰素的释放，细胞毒作用)。但在缺乏CD3抗体信号时，CD137分子的刺激并不能改变T细胞的功能，表明CD137只是一种协同共刺激分子。
CD137刺激T细胞后的生物活性是由NF-κB和PI3K/ERK1/2两个独立的生理功能信号介导的。NF-κB信号引发抑制凋亡分子Bcl-XL的表达，从而提高了生存能力，而PI3K和ERK1/2的信号则具体负责CD137介导的细胞周期进程。CD137的激活可抑制活化诱导的细胞死亡，该作用最初由Hurtado等人于体外证明，后来在体内实验中证实，CD137的单克隆抗体(mAbs)可通过防止克隆缺失产生长期存活的超抗原激活型CD8+T细胞。这两份研究报告表明，在不同的实验条件下，CD137的信号即可调节克隆扩增还可调节CD8+T细胞的存活。协同刺激相关的CD8+T细胞内Bcl-XL增加可减少细胞凋亡，而Bcl-2的表达维持不变。由于PDTC(NF-κB特异性阻断剂)可抑制CD137介导的Bcl-XL上调，4-1BB引起的抑制凋亡基因Bcl-XL和bfl-1的表达上调是由NF-κb激活介导的。另一方面，激活T细胞的克隆扩增似乎是由细胞周期蛋白D2，D3和E表达增加和p27Kip1蛋白的表达下调介导的。这种效应既可以IL-2依赖的模式也可以IL-2非依赖的模式发生。
总之，CD137的刺激能协同CD28进一步活化T细胞，维持CD8+T细胞的增值、生存和效应功能。虽然CD4+和CD8+T细胞均可对CD137的刺激做出应答，但CD137可优先介导CD8+T细胞的免疫活性。基于CD137协同刺激信号在CD8+T细胞活化和效应中所起的重要作用，利用CD137/CD137L系统便可为肿瘤免疫治疗的设计提供了新的作用途径。
CD137靶向免疫治疗在癌症治疗方面的作用最初是由在体内利用激活型鼠源CD137单抗的疗效研究表明的。Melero等研究指出，激活型鼠源CD137单抗可以消除已经建立的P815肿瘤实体瘤，对于弱肿瘤免疫原性的AG104A肉瘤也有明显的效果，这种抗肿瘤的效果具有完全治愈性，并对于下次肿瘤的攻击有记忆性，证明有长期的抗肿瘤免疫反应存在。自那时起，便有人开始使用CD137抗体来证明这种抗肿瘤疗法的可行性。目前，美国BMS公司针对CD137的单克隆抗体在一期临床试验中显示出良好的耐受性和较好的初步疗效，已进入二期临床试验用于治疗转移性黑色素瘤。
与激活型CD137抗体的抗肿瘤效果相一致的是，CD137L可激发CTL效应和抗肿瘤反应。研究显示，将CD137L基因转染到小鼠癌细胞中可产生肿瘤排斥反应，说明产生有效免疫反应是需要协同刺激的。CD137L可表达于多种恶性肿瘤细胞表面，表明表达配体的肿瘤细胞能向T细胞递呈一个协同刺激信号，这个信号又可引起IFN-α与IL-2的释放。该效应是与肿瘤中CD137L的表达呈正相关的，但肿瘤细胞所表达的CD137L能否使这些细胞对激活型CD137抗体更加敏感尚有待研究。
CD137L-/-鼠很好的说明了CD137/CD137L系统在T细胞对病毒和肿瘤免疫应答中所起的重要作用。使用CD137或CD137L缺陷型鼠的研究表明CD137的协同刺激作用对移植物抗宿主病、T细胞的抗病毒融细胞性应答中起重要作用。CD137缺陷型鼠增强了T细胞的增殖能力，但减少了细胞因子的生产能力和细胞毒性T细胞的活性。最近研究表明，与对照组小鼠相比，CD137-/-鼠肿瘤转移频率(4倍)提高。因此，通过使用激活型CD137抗体恢复CD137的信号是提高细胞对病毒和肿瘤免疫应答的一种可行方法。
Ewing’s肉瘤患者体内循环T细胞表达MHC分子，且抗肿瘤效应T细胞属于记忆性CD3+CD8+T细胞，而且是CD28-/CD137+T细胞。自身Ewing’s肉瘤细胞表达CD137L，后者提供的协同刺激可诱导T细胞活化与增殖，这是通过CD3mAb和CD137L mAb刺激外周血(PBL)实现的，而不是CD3mAb和CD28mAb刺激PBL产生。前者可以扩增那些控制原始肿瘤细胞生长的T细胞，而且能控制肿瘤的转移，而后者则无此作用。Zhang等在实验中观察到肿瘤进行性生长与肿瘤自身4-1BBL诱导抗肿瘤免疫同时存在。这进一步证实了CD137L介导的协同刺激在扩增肿瘤特异性效应CTL并用于过继性免疫治疗中潜在的作用。除了在癌症免疫方面的作用外，CD137在自身免疫性疾病和对HIV的作用也有报道。
综上所述，近年已利用CD137mAb、CD137L基因转染以及与其它分子协同，用于肿瘤的免疫治疗并显示出良好的前景，但它们都存在成本高、副作用明显的问题，难以实现产业化。鉴于CD137/CD137L在免疫应答和免疫调节中所起的特殊作用，采用基因工程方法，克隆和表达人具有CD137L功能的可溶性重组蛋白，研究其在调节机体免疫力、细胞增殖和机体细胞因子的合成与分泌中的作用，以及在抗病毒、抗肿瘤方面的应用，可为肿瘤的过继免疫治疗提供新的策略。
发明内容
本发明的目的是提供一种具有CD137L功能的蛋白或多肽。
本发明的另一个目的是提供编码上述具有CD137L功能的蛋白或多肽的基因。
本发明还有一个目的是提供上述具有CD137L功能的蛋白或多肽的制备方法。
本发明再有一个目的是提供上述具有CD137L功能的蛋白或多肽及其基因在制药中的应用。
本发明的目的是通过下列技术方案实现的：
一种编码具有CD137L功能的蛋白或多肽的基因，它具有SEQ ID NO.1至SEQ IDNO.16所示之一的核苷酸序列。
一种具有CD137L功能的蛋白或多肽，该蛋白或多肽为CD137L胞外区蛋白(NM_003811，第147位至第773位核苷酸)和His tag标签通过0-3个氨基酸的连接肽融合而成，优选1-3个氨基酸的连接肽。
所述的具有CD137L功能的蛋白或多肽，其中所述氨基酸连接肽选自下列任意一种：Leu-Glu，Arg-Leu-Glu，Ile-Leu-Glu，Pro-Leu-Glu，Cys，Met，Tyr，Lys，Leu-Cys，Ile-Lys，Asp-Lys，Thr-Glu，Ile-Tyr-Met，Thr-Leu-Val，Thr-Leu-Val和Asp-Ser-Lys。
所述的具有CD137L功能的蛋白或多肽，它具有SEQ ID NO.17至SEQ ID NO.32所示之一的氨基酸序列。
所述的具有CD137L功能的蛋白或多肽的制备方法，包括下列步骤：
将编码上述具有CD137L功能的蛋白或多肽的任一核苷酸序列(优选SEQ ID NO.1至SEQ ID NO.16所示任一的核苷酸序列)插入表达载体，转化大肠杆菌，获得能够表达具有CD137L功能的蛋白或多肽的基因工程菌，通过液体培养基因工程菌，经亲和层析纯化制得具有CD137L功能的蛋白或多肽。
所述的具有CD137L功能的蛋白或多肽的基因在制备调节机体免疫力、细胞增殖和机体细胞因子的合成与分泌的药物中的应用。
所述的具有CD137L功能的蛋白或多肽在制备调节机体免疫力、细胞增殖和机体细胞因子的合成与分泌的药物中的应用。
具体地说，本发明的一种编码具有CD137L功能的蛋白或多肽基因是以B淋巴瘤Raji细胞系mRNA为模板，采用一系列人CD137L胞外区基因片段的特异性引物，通过RT-PCR技术获得CD137L胞外区基因文库。该文库由CD137L胞外区基因片段通过不同数目的氨基酸连接肽的基因片段与His tag标签通过不同方式融合组成(图1)。
本发明还构建了含有上述编码具有CD137L功能的蛋白或多肽基因的载体，这些载体的构建方法是按照常规方法，将通过RT-PCR获得的CD137L胞外区基因经NdeI和XhoI双酶切后，连接入相应载体的相应酶切位点之间。含有上述具有CD137L功能的蛋白或多肽基因的大肠杆菌表达载体优先由本发明合成的编码具有CD137L功能的重组蛋白的基因与大肠杆菌表达载体pET22b构建而成。
本发明还提供了利用上述含大肠杆菌重组表达载体的大肠杆菌重组株生产具有CD137L功能的蛋白或多肽的方法，该方法是将菌种进行培养发酵并经低温诱导使其高效表达CD137L重组蛋白，再收集菌体，破细胞离心得到的上清经Ni亲和柱层析一步纯化获得具有CD137L功能的重组蛋白，SDS-PAGE电泳纯度大于95％。
本发明对具有CD137L功能的重组蛋白的结构鉴定是用Invitrogen公司的InVisionHis-tag In-gel Stain检测所纯化的蛋白，结果证明均具有His-tag标签。对上述纯化的具有CD137L功能的蛋白或多肽蛋白进行细胞ELISA实验显示，所述重组蛋白与CD137表达阳性的人胃癌细胞Bgc803有较强的亲和力，并且具有剂量依赖性。对所获重组蛋白的体外生物学活性进行了初步功能鉴定和分析，经实验验证，融合蛋白质能保持CD137L的生物活性，可协同抗CD3和抗CD28单克隆抗体刺激T细胞的增殖，提高细胞因子IL-2的释放。
本发明的有益效果：
本发明表明，采用原核表达系统表达产生由CD137L胞外区片段与His-tag标签通过融合组成的CD137L重组蛋白，同样可以产生具有CD137L的结构和生理功能。利用本发明生产CD137L重组蛋白(具有CD137L功能的蛋白或多肽)，具有表达量效率高、表达量大、表达周期短，易于纯化等优点，并且，本发明提供的具有CD137L功能的蛋白或多肽与CD137表达阳性的人胃癌细胞Bgc803有较强的亲和力，并且具有剂量依赖性，也可协同抗CD3和抗CD28单克隆抗体刺激T细胞的增殖，提高细胞因子IL-2的释放。因此，本发明为大规模生产CD137L蛋白提供了新的、安全的途径，为进一步研究和开发成为新一代抗肿瘤药物奠定了扎实的基础，在制药行业具有广阔的应用前景。
附图说明
图1为具有CD137L功能的重组蛋白结构示意图。X为0-3个氨基酸的连接肽，可为如下所示之一的氨基酸组合：Leu-Glu，Arg-Leu-Glu，Ile-Leu-Glu，Pro-Leu-Glu，Cys，Met，Tyr，Lys，Leu-Cys，Ile-Lys，Asp-Lys，Thr-Glu，Ile-Tyr-Met，Thr-Leu-Val，Thr-Leu-Val和Asp-Ser-Lys。
图2为SDS-PAGE分析不同IPTG浓度对代表性CD137L重组蛋白表达的影响。泳道1-6为0，0.1，0.2，0.3，0.4和0.5mM IPTG诱导目的蛋白表达(SEQ ID NO.20)的上清；泳道M为蛋白标准分子量。
图3为SDS-PAGE分析Ni柱纯化的代表性具有CD137L功能的重组蛋白。泳道1-6所对应的目的蛋白为SEQ ID NO.17，19，20，21，25和30；泳道M为蛋白标准分子量。
图4为SDS-PAGE分析2种代表性CD137L重组蛋白的His-tag标签。图4A为考马斯亮蓝染色，图4B为Invision His-tag in-gel stain染色。泳道1和2分别对应的目的蛋白为SEQ ID NO.19和20；泳道M为蛋白标准分子量。
图5为细胞ELISA法测定3种代表性具有CD137L功能的重组蛋白与CD137的亲和力。1，2和3分别为SEQ ID NO.17，19和20。
图6为4种代表性具有CD137L功能的重组蛋白对人外周血T淋巴细胞增殖的影响。1，2，3和4分别为SEQ ID NO.17，19，20和25。
图7为6种代表性具有CD137L功能的重组蛋白CD137L类似物对人外周血T淋巴细胞IL-2释放的影响。TBlank为未加任何抗体刺激的T细胞空白组；T+CD3+CD28为抗CD3和CD28单克隆抗体联合刺激组；1-6分别为SEQ ID NO.17，19，20，21，25和30联合抗CD3和CD28单克隆抗体刺激组。
图2、3、4中的“CD137L”指本发明“具有CD137L功能的重组蛋白”。
具体实施方式
下面结合附图及实施例对本发明作进一步描述：
实验材料和试剂：
B淋巴瘤Raji细胞系和人胃癌Bgc803细胞系购自上海科学院细胞中心购得，pET22b(+)和pET28a(+)由Novagen公司购得，Top10和BL21(DE3)菌株由Invitrogen公司购得。pMD18-T载体(货号：D103A)、逆转录酶、T4DNA连接酶和限制性内切酶均购自TaKaRa公司。引物的合成和核苷酸序列测序由上海英骏生物技术有限公司完成。纯化所用的Ni亲和层析柱购自GE Healthcare公司。抗CD3和抗CD28单克隆抗体购自Santa Cruz公司。人T细胞富集试剂盒购自Stem cell公司。人IL-2ELISA试剂盒购自Bender MedSystems公司。其它试剂均为国产分析纯。
实施例1：具有CD137L功能的重组蛋白基因表达载体的构建
共设计了16种具有CD137L功能的蛋白或多肽，其核苷酸序列为SEQ ID NO.1-16，对应的氨基酸序列为SEQ ID NO.17-32。该蛋白或多肽为CD137L胞外区蛋白和His-tag标签通过0-3个氨基酸的连接肽融合而成，优选1-3个氨基酸的连接肽，其中His-tag标签在N端的有4种，在C端的有12种(图1)。该氨基酸连接肽可为如下所示之一的氨基酸组合：Leu-Glu，Arg-Leu-Glu，Ile-Leu-Glu，Pro-Leu-Glu，Cys，Met，Tyr，Lys，Leu-Cys，Ile-Lys，Asp-Lys，Thr-Glu，Ile-Tyr-Met，Thr-Leu-Val和Asp-Ser-Lys。
SEQ ID NO.1-4的上游引物为：5’-TCATATG GCCGTCTTCCTCGCCT-3’；
SEQ ID NO.1-4的下游引物分别为：
5’-T CTCGAG TTCCGACCTCGGTGAAG-3’，
5’-T CTCGAG ACG TTCCGACCTCGGTGAAG-3’，
5’-T CTCGAG GAT TTCCGACCTCGGTGAAG-3’
5’-T CTCGAG CGG TTCCGACCTCGGTGAAG-3’。
SEQ ID NO.5-16的下游引物为：
5’-T CTCGAG TTA TTCCGACCTCGGTGAAGGGAGTCCG-3’；
SEQ ID NO.5-16的上游引物分别为：
5’-T CATATG CATCATCACCATCATCAT GCCGTCTTCCTCGCCT-3’，
5’-T CATATG CATCATCATCACCATCAT TGC GCCGTCTTCCTCGCCT-3’，
5’-T CATATG CATCATCATCACCATCAT ATG GCCGTCTTCCTCGCCT-3’，
5’-T CATATG CATCATCATCACCATCAT TAC GCCGTCTTCCTCGCCT-3’，
5’-T CATATG CATCATCATCACCATCAT AAG GCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCACCATTTATGC GCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCACCATATTAAGGCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCACCATGATAAGGCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCACCATACGGAAGCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCATCACATCTACATGGCCGTCTTCCTCGCCT-3’，
5’-TCATATGCATCATCATCATCATCACACTCTTGTTGCCGTCTTCCTCGCCT-3’
5’-TCATATGCATCATCATCATCATCACGATTCTAAGGCCGTCTTCCTCGCCT-3’。
所有上游引物中均含有NdeI酶切位点，下游引物中均含有XhoI酶切位点。
从B淋巴瘤Raji细胞系中提取mRNA，并用Dnase I(无RNA酶)处理去除可能残余的基因组DNA。在EP管(无RNA酶)中加入2μg总RNA、0.5μg Oligo(dT)18以及适量DEPC-ddH2O(使终体积为25μL)，70℃水浴5min热变性后立即置于冰水浴中。加入：5μL M-MLV 5×反应缓冲液、1.25μL dNTP(每10mM)、40U RNAsin、200UM-MLV逆转录酶，37℃温育1.5h，95℃加热5min灭活逆转录酶，第一链cDNA可于-20℃下保存3个月。以反转录的cDNA第一链为模板，分别利用上述设计的各对引物进行PCR扩增。反应体系如下：
10PCR Buffer 3μl
dNTP 2μl
上游引物 2μl
下游引物 2μl
质粒 1μl
ddH2O 20μl
Taq 0.5μl
总体积 30μl
将反应混合物置于PCR扩增仪，94℃5min；然后在94℃1min，58℃1min，72℃1min循环，共29个循环，最后72℃延伸10min。将该PCR产物回收后，与pMD-18T载体连接。连接体系为pMD-18T 1μL，目的片段1μL，无菌水3μL，溶液I 5μL(该试剂盒的货号：D103A，购自TaKaRa公司)，连接条件为16℃连接16h。转化大肠杆菌TOP10。在含有氨苄青霉素的LB固体培养基上培养12h后挑取单克隆，提取质粒后用限制性内切酶NdeI和XhoI进行双酶切鉴定，对酶切验证正确的菌落送交上海英骏生物技术有限公司测序，以确定基因序列的正确性。将测序正确的目的片段与表达载体pET22b连接，连接体系为T4连接酶缓冲液1μL，pET22b 2μL，目的基因6μL，T4连接酶1μL，连接条件为16℃连接16h。双酶切验证后，转化大肠杆菌BL21(DE3)。
实施例2：具有CD137L功能的重组蛋白的表达
在超净台中，向含100μg/mL氨苄青霉素的LB培养基中分别加入1mL上述保存含有目的基因质粒的大肠杆菌BL21(DE3)菌种至100mL菌液中，置于摇床上，37℃，250rpm条件下过夜培养。将活化后的菌种转接到新的灭菌的LB培养基中，于OD600值为0.9时加入IPTG，使同一菌种终浓度分别为0，0.1，0.2，0.3，0.4和0.5mM，15℃培养16h后离心，收集菌体。菌体按湿重1g加10mL PBS吹悬，加入高压破细胞仪中破碎细胞，收集流出液，4℃，12000rpm离心30min，弃沉淀，留上清做SDS-PAGE分析。
SDS-PAGE具体操作如下：
1)每管各取3ml于EP管中，12000rpm×1.2min离心，弃上清。用1ml ddH2O洗涤，离心12000rpm×1min，弃上清，再用100μl ddH2O重悬。
2)取20μl重悬菌液加5μl 5×SDS-PAGE上样缓冲液，沸水浴10min，12000rpm×10min离心。
3)参照《分子克隆手册》灌制SDS-PAGE胶。5％积层胶，12％分离胶，1×Tris-Gly电泳缓冲液。
4)各取20μl上清液上样，电泳。电泳结束后取下胶，考马斯亮蓝染色1-2h。取出后用去离子水清洗三次，置脱色液中浸泡过夜。
电泳结果如图2所示，IPTG浓度在0.1mM时即可较好地诱导工程菌表达目的蛋白(以SEQ ID NO.20为例)，随IPTG浓度的增高目的蛋白表达无显著性差异。
实施例3：具有CD137L功能的重组蛋白的纯化
采用上述方法收集高压破碎后所得的上清，用0.45μm滤膜过滤。采用GE公司的Ni Sepharose High Performance层析柱纯化目的蛋白，分别用含20mM，50mM，100mM和400mM咪唑浓度的缓冲液进行阶段洗脱，流速为2ml/min，收集各阶段洗脱峰，SDS-PAGE检测蛋白纯化效果。实验结果显示，大部分杂蛋白随着缓冲液咪唑浓度的提高逐渐被洗脱下来，当咪唑浓度上升到50mM时，重组融合蛋白开始被洗脱下来；当咪唑浓度上升到100mM时，仍有一些杂蛋白被同时洗脱；而当咪唑浓度达到400mM，在25kDa处可得到一特异条带，符合目的蛋白的理论推算的分子量，其纯度达95％以上。所纯化后的代表性目的蛋白的电泳结果见图3，泳道1-6所对应的目的蛋白为SEQ IDNO.17，19，20，21，25和30。
实施例4：具有CD137L功能的重组蛋白的His-tag检测
用Invitrogen公司的InVision His-tag In-gel Stain检测目的蛋白的His-tag标签。该试剂利用荧光染料交联带有Ni2+的NTA，其中的Ni2+可以高度亲和His-tag，所以可以直接染色PAGE胶，而特异地将样品中带有His-Tag的条带显示出来。SDS-PAGE电泳结束后，先用该染料染色，紫外照射下拍照，之后再用考马斯亮蓝染色。实验结果显示，所纯化的蛋白均具有His-tag标签(图4)。
实施例5：细胞ELISA法测定CD137L与CD137的亲和力
培养人胃癌Bgc803细胞系(CD137表达阳性细胞)，加入96孔细胞培养板，贴壁后，3％的牛血清白蛋白(BSA)37℃封闭1h。用RMPI 1640稀释具有CD137L功能的重组蛋白样品加入96孔板孵育2h，使其终浓度分别为30，15，7.5，3.75和1.875μg/mL，用PBS洗5次；加入稀释的HRP标记的鼠抗His-tag抗体，37℃孵育1h，PBS洗5次；然后用邻苯二胺(OPD)显色，在酶标仪上测定波长为490nm的OD值。结果表明纯化后的具有CD137L功能的重组蛋白可与Bgc803细胞结合，并且具有剂量依赖性(图5)。
实施例6：具有CD137L功能的重组蛋白对人外周血T淋巴细胞增殖的影响按照Stemcell公司的Human T Cell Enrichment Cocktail试剂盒操作，从人外周血中分离、获得纯化的T细胞群体。人CD3单克隆抗体按5μg/mL包被96孔板，4℃过夜，第2天加入纯化的人外周血T淋巴细胞(每孔104/100μL)，共分成4个实验组：①未加任何抗体刺激的T细胞空白组(T cell)；②抗CD3单克隆抗体(5μg/mL)刺激组(CD3)；③抗CD3和抗CD28单克隆抗体(5μg/mL)联合刺激组(CD3+CD28)；④4种代表性CD137L蛋白(SEQ ID NO.17-20)分别联合抗CD3和抗CD28单克隆抗体刺激组(CD3+CD28+CD137L)每组置3个复孔。培养68h后，每孔加20uL MTT(5mg/mL)，4h后加酸化的SDS溶解，测OD值。实验结果如图6所示，4种代表性CD137L蛋白在终浓度为2μg/mL时对T细胞的增殖显著高于“CD3+CD28”协同刺激组，证明CD137L和CD28具有协同效应。由此表明，所获得的CD137L蛋白具有良好的协同激发T细胞增殖的生物学功能。
实施例7：CD137L重组蛋白对人外周血T淋巴细胞IL-2释放的影响
按上述方法从人外周血中分离、获得纯化的T细胞群体。人抗CD3单克隆抗体按7.5μg/mL包被96孔板，4℃过夜。第2天加入纯化的人外周血T淋巴细胞(每孔2×104/100μL)，共分成8个实验组：①未加任何抗体刺激的T细胞空白组；②CD3和CD28单克隆抗体(2.5μg/mL)联合刺激组(T+CD3+CD28)；③1lμg/mL或10μg/mL 6种代表性CD137L重组蛋白(SEQ ID NO.17-22)分别联合抗CD3和CD28单克隆抗体刺激组。培养48h后，离心取上清，按照人IL-2ELISA试剂盒操作，检测细胞因子IL-2的浓度。实验结果如图7所示，1和10μg/mL的CD137L蛋白均可显著协同抗CD3和CD28单克隆抗体刺激激活的T细胞，提高培养液中IL-2的释放，但T细胞激活程度在各组间有较大差异。
序列表
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ttggacctgc ggcagggcat gtttgcgcag ctggtggccc aaaatgttct gctgatcgat 180
gggcccctga gctggtacag tgacccaggc ctggcaggcg tgtccctgac ggggggcctg 240
agctacaaag aggacacgaa ggagctggtg gtggccaagg ctggagtcta ctatgtcttc 300
tttcaactag agctgcggcg cgtggtggcc ggcgagggct caggctccgt ttcacttgcg 360
ctgcacctgc agccactgcg ctctgctgct ggggccgccg ccctggcttt gaccgtggac 420
ctgccacccg cctcctccga ggctcggaac tcggccttcg gtttccaggg ccgcttgctg 480
cacctgagtg ccggccagcg cctgggcgtc catcttcaca ctgaggccag ggcacgccat 540
gcctggcagc ttacccaggg cgccacagtc ttgggactct tccgggtgac ccccgaaatc 600
ccagccggac tcccttcacc gaggtcggaa ctcgagcacc accaccacca ccactga 657
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ttggacctgc ggcagggcat gtttgcgcag ctggtggccc aaaatgttct gctgatcgat 180
gggcccctga gctggtacag tgacccaggc ctggcaggcg tgtccctgac ggggggcctg 240
agctacaaag aggacacgaa ggagctggtg gtggccaagg ctggagtcta ctatgtcttc 300
tttcaactag agctgcggcg cgtggtggcc ggcgagggct caggctccgt ttcacttgcg 360
ctgcacctgc agccactgcg ctctgctgct ggggccgccg ccctggcttt gaccgtggac 420
ctgccacccg cctcctccga ggctcggaac tcggccttcg gtttccaggg ccgcttgctg 480
cacctgagtg ccggccagcg cctgggcgtc catcttcaca ctgaggccag ggcacgccat 540
gcctggcagc ttacccaggg cgccacagtc ttgggactct tccgggtgac ccccgaaatc 600
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gggcccctga gctggtacag tgacccaggc ctggcaggcg tgtccctgac ggggggcctg 240
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gcctggcagc ttacccaggg cgccacagtc ttgggactct tccgggtgac ccccgaaatc 600
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ttggacctgc ggcagggcat gtttgcgcag ctggtggccc aaaatgttct gctgatcgat 180
gggcccctga gctggtacag tgacccaggc ctggcaggcg tgtccctgac ggggggcctg 240
agctacaaag aggacacgaa ggagctggtg gtggccaagg ctggagtcta ctatgtcttc 300
tttcaactag agctgcggcg cgtggtggcc ggcgagggct caggctccgt ttcacttgcg 360
ctgcacctgc agccactgcg ctctgctgct ggggccgccg ccctggcttt gaccgtggac 420
ctgccacccg cctcctccga ggctcggaac tcggccttcg gtttccaggg ccgcttgctg 480
cacctgagtg ccggccagcg cctgggcgtc catcttcaca ctgaggccag ggcacgccat 540
gcctggcagc ttacccaggg cgccacagtc ttgggactct tccgggtgac ccccgaaatc 600
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gacgatcccg ccggcctctt ggacctgcgg cagggcatgt ttgcgcagct ggtggcccaa 180
aatgttctgc tgatcgatgg gcccctgagc tggtacagtg acccaggcct ggcaggcgtg 240
tccctgacgg ggggcctgag ctacaaagag gacacgaagg agctggtggt ggccaaggct 300
ggagtctact atgtcttctt tcaactagag ctgcggcgcg tggtggccgg cgagggctca 360
ggctccgttt cacttgcgct gcacctgcag ccactgcgct ctgctgctgg ggccgccgcc 420
ctggctttga ccgtggacct gccacccgcc tcctccgagg ctcggaactc ggccttcggt 480
ttccagggcc gcttgctgca cctgagcgcc ggccagcgcc tgggcgtcca tcttcacact 540
gaggccaggg cacgccatgc ctggcagctt acccagggcg ccacagtctt gggactcttc 600
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cccgacgatc ccgccggcct cttggacctg cggcagggca tgtttgcgca gctggtggcc 180
caaaatgttc tgctgatcga tgggcccctg agctggtaca gtgacccagg cctggcaggc 240
gtgtccctga cggggggcct gagctacaaa gaggacacga aggagctggt ggtggccaag 300
gctggagtct actatgtctt ctttcaacta gagctgcggc gcgtggtggc cggcgagggc 360
tcaggctccg tttcacttgc gctgcacctg cagccactgc gctctgctgc tggggccgcc 420
gccctggctt tgaccgtgga cctgccaccc gcctcctccg aggctcggaa ctcggccttc 480
ggtttccagg gccgcttgct gcacctgagt gccggccagc gcctgggcgt ccatcttcac 540
actgaggcca gggcacgcca tgcctggcag cttacccagg gcgccacagt cttgggactc 600
ttccgggtga cccccgaaat cccagccgga ctcccttcac cgaggtcgga ataa 654
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atgcatcatc atcaccatca tatggccgtc ttcctcgcct gcccctgggc cgtgtccggg 60
gctcgcgcct cgcccggctc cgcggccagc ccgagactcc gcgagggtcc cgagctttcg 120
cccgacgatc ccgccggcct cttggacctg cggcagggca tgtttgcgca gctggtggcc 180
caaaatgttc tgctgatcga tgggcccctg agctggtaca gtgacccagg cctggcaggc 240
gtgtccctga cggggggcct gagctacaaa gaggacacga aggagctggt ggtggccaag 300
gctggagtct actatgtctt ctttcaacta gagctgcggc gcgtggtggc cggcgagggc 360
tcaggctccg tttcacttgc gctgcacctg cagccactgc gctctgctgc tggggccgcc 420
gccctggctt tgaccgtgga cctgccaccc gcctcctccg aggctcggaa ctcggccttc 480
ggtttccagg gccgcttgct gcacctgagt gccggccagc gcctgggcgt ccatcttcac 540
actgaggcca gggcacgcca tgcctggcag cttacccagg gcgccacagt cttgggactc 600
ttccgggtga cccccgaaat cccagccgga ctcccttcac cgaggtcgga ataa 654
<210>8
<211>654
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>8
atgcatcatc atcaccatca ttacgccgtc ttcctcgcct gcccctgggc cgtgtccggg 60
gctcgcgcct cgcccggctc cgcggccagc ccgagactcc gcgagggtcc cgagctttcg 120
cccgacgatc ccgccggcct cttggacctg cggcagggca tgtttgcgca gctggtggcc 180
caaaatgttc tgctgatcga tgggcccctg agctggtaca gtgacccagg cctggcaggc 240
gtgtccctga cggggggcct gagctacaaa gaggacacga aggagctggt ggtggccaag 300
gctggagtct actatgtctt ctttcaacta gagctgcggc gcgtggtggc cggcgagggc 360
tcaggctccg tttcacttgc gctgcacctg cagccactgc gctctgctgc tggggccgcc 420
gccctggctt tgaccgtgga cctgccaccc gcctcctccg aggctcggaa ctcggccttc 480
ggtttccagg gccgcttgct gcacctgagt gccggccagc gcctgggcgt ccatcttcac 540
actgaggcca gggcacgcca tgcctggcag cttacccagg gcgccacagt cttgggactc 600
ttccgggtga cccccgaaat cccagccgga ctcccttcac cgaggtcgga ataa 654
<210>9
<211>654
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>9
atgcatcatc atcaccatca taaggccgtc ttcctcgcct gcccctgggc cgtgtccggg 60
gctcgcgcct cgcccggctc cgcggccagc ccgagactcc gcgagggtcc cgagctttcg 120
cccgacgatc ccgccggcct cttggacctg cggcagggca tgtttgcgca gctggtggcc 180
caaaatgttc tgctgatcga tgggcccctg agctggtaca gtgacccagg cctggcaggc 240
gtgtccctga cggggggcct gagctacaaa gaggacacga aggagctggt ggtggccaag 300
gctggagtct actatgtctt ctttcaacta gagctgcggc gcgtggtggc cggcgagggc 360
tcaggctccg tttcacttgc gctgcacctg cagccactgc gctctgctgc tggggccgcc 420
gccctggctt tgaccgtgga cctgccaccc gcctcctccg aggctcggaa ctcggccttc 480
ggtttccagg gccgcttgct gcacctgagt gccggccagc gcctgggcgt ccatcttcac 540
actgaggcca gggcacgcca tgcctggcag cttacccagg gcgccacagt cttgggactc 600
ttccgggtga cccccgaaat cccagccgga ctcccttcac cgaggtcgga ataa 654
<210>10
<211>657
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>10
atgcatcatc atcatcacca tttatgcgcc gtcttcctcg cctgcccctg ggccgtgtcc 60
ggggctcgcg cctcgcccgg ctccgcggcc agcccgagac tccgcgaggg tcccgagctt 120
tcgcccgacg atcccgccgg cctcttggac ctgcggcagg gcatgtttgc gcagctggtg 180
gcccaaaatg ttctgctgat cgatgggccc ctgagctggt acagtgaccc aggcctggca 240
ggcgtgtccc tgacgggggg cctgagctac aaagaggaca cgaaggagct ggtggtggcc 300
aaggctggag tctactatgt cttctttcaa ctagagctgc ggcgcgtggt ggccggcgag 360
ggctcaggct ccgtttcact tgcgctgcac ctgcagccac tgcgctctgc tgctggggcc 420
gccgccctgg ctttgaccgt ggacctgcca cccgcctcct ccgaggctcg gaactcggcc 480
ttcggtttcc agggccgctt gctgcacctg agtgccggcc agcgcctggg cgtccatctt 540
cacactgagg ccagggcacg ccatgcctgg cagcttaccc agggcgccac agtcttggga 600
ctcttccggg tgacccccga aatcccagcc ggactccctt caccgaggtc ggaataa 657
<210>11
<211>657
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>11
atgcatcatc atcatcacca tattaaggcc gtcttcctcg cctgcccctg ggccgtgtcc 60
ggggctcgcg cctcgcccgg ctccgcggcc agcccgagac tccgcgaggg tcccgagctt 120
tcgcccgacg atcccgccgg cctcttggac ctgcggcagg gcatgtttgc gcagctggtg 180
gcccaaaatg ttctgctgat cgatgggccc ctgagctggt acagtgaccc aggcctggca 240
ggcgtgtccc tgacgggggg cctgagctac aaagaggaca cgaaggagct ggtggtggcc 300
aaggctggag tctactatgt cttctttcaa ctagagctgc ggcgcgtggt ggccggcgag 360
ggctcaggct ccgtttcact tgcgctgcac ctgcagccac tgcgctctgc tgctggggcc 420
gccgccctgg ctttgaccgt ggacctgcca cccgcctcct ccgaggctcg gaactcggcc 480
ttcggtttcc agggccgctt gctgcacctg agtgccggcc agcgcctggg cgtccatctt 540
cacactgagg ccagggcacg ccatgcctgg cagcttaccc agggcgccac agtcttggga 600
ctcttccggg tgacccccga aatcccagcc ggactccctt caccgaggtc ggaataa 657
<210>12
<211>657
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>12
atgcatcatc atcatcacca tgataaggcc gtcttcctcg cctgcccctg ggccgtgtcc 60
ggggctcgcg cctcgcccgg ctccgcggcc agcccgagac tccgcgaggg tcccgagctt 120
tcgcccgacg atcccgccgg cctcttggac ctgcggcagg gcatgtttgc gcagctggtg 180
gcccaaaatg ttctgctgat cgatgggccc ctgagctggt acagtgaccc aggcctggca 240
ggcgtgtccc tgacgggggg cctgagctac aaagaggaca cgaaggagct ggtggtggcc 300
aaggctggag tctactatgt cttctttcaa ctagagctgc ggcgcgtggt ggccggcgag 360
ggctcaggct ccgtttcact tgcgctgcac ctgcagccac tgcgctctgc tgctggggcc 420
gccgccctgg ctttgaccgt ggacctgcca cccgcctcct ccgaggctcg gaactcggcc 480
ttcggtttcc agggccgctt gctgcacctg agtgccggcc agcgcctggg cgtccatctt 540
cacactgagg ccagggcacg ccatgcctgg cagcttaccc agggcgccac agtcttggga 600
ctcttccggg tgacccccga aatcccagcc ggactccctt caccgaggtc ggaataa 657
<210>13
<211>657
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>13
atgcatcatc atcatcacca tacggaagcc gtcttcctcg cctgcccctg ggccgtgtcc 60
ggggctcgcg cctcgcccgg ctccgcggcc agcccgagac tccgcgaggg tcccgagctt 120
tcgcccgacg atcccgccgg cctcttggac ctgcggcagg gcatgtttgc gcagctggtg 180
gcccaaaatg ttctgctgat cgatgggccc ctgagctggt acagtgaccc aggcctggca 240
ggcgtgtccc tgacgggggg cctgagctac aaagaggaca cgaaggagct ggtggtggcc 300
aaggctggag tctactatgt cttctttcaa ctagagctgc ggcgcgtggt ggccggcgag 360
ggctcaggct ccgtttcact tgcgctgcac ctgcagccac tgcgctctgc tgctggggcc 420
gccgccctgg ctttgaccgt ggacctgcca cccgcctcct ccgaggctcg gaactcggcc 480
ttcggtttcc agggccgctt gctgcacctg agtgccggcc agcgcctggg cgtccatctt 540
cacactgagg ccagggcacg ccatgcctgg cagcttaccc agggcgccac agtcttggga 600
ctcttccggg tgacccccga aatcccagcc ggactccctt caccgaggtc ggaataa 657
<210>14
<211>660
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>14
atgcatcatc atcatcatca catctacatg gccgtcttcc tcgcctgccc ctgggccgtg 60
tccggggctc gcgcctcgcc cggctccgcg gccagcccga gactccgcga gggtcccgag 120
ctttcgcccg acgatcccgc cggcctcttg gacctgcggc agggcatgtt tgcgcagctg 180
gtggcccaaa atgttctgct gatcgatggg cccctgagct ggtacagtga cccaggcctg 240
gcaggcgtgt ccctgacggg gggcctgagc tacaaagagg acacgaagga gctggtggtg 300
gccaaggctg gagtctacta tgtcttcttt caactagagc tgcggcgcgt ggtggccggc 360
gagggctcag gctccgtttc acttgcgctg cacctgcagc cactgcgctc tgctgctggg 420
gccgccgccc tggctttgac cgtggacctg ccacccgcct cctccgaggc tcggaactcg 480
gccttcggtt tccagggccg cttgctgcac ctgagtgccg gccagcgcct gggcgtccat 540
cttcacactg aggccagggc acgccatgcc tggcagctta cccagggcgc cacagtcttg 600
ggactcttcc gggtgacccc cgaaatccca gccggactcc cttcaccgag gtcggaataa 660
<210>15
<211>660
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>15
atgcatcatc atcatcatca cactcttgtt gccgtcttcc tcgcctgccc ctgggccgtg 60
tccggggctc gcgcctcgcc cggctccgcg gccagcccga gactccgcga gggtcccgag 120
ctttcgcccg acgatcccgc cggcctcttg gacctgcggc agggcatgtt tgcgcagctg 180
gtggcccaaa atgttctgct gatcgatggg cccctgagct ggtacagtga cccaggcctg 240
gcaggcgtgt ccctgacggg gggcctgagc tacaaagagg acacgaagga gctggtggtg 300
gccaaggctg gagtctacta tgtcttcttt caactagagc tgcggcgcgt ggtggccggc 360
gagggctcag gctccgtttc acttgcgctg cacctgcagc cactgcgctc tgctgctggg 420
gccgccgccc tggctttgac cgtggacctg ccacccgcct cctccgaggc tcggaactcg 480
gccttcggtt tccagggccg cttgctgcac ctgagtgccg gccagcgcct gggcgtccat 540
cttcacactg aggccagggc acgccatgcc tggcagctta cccagggcgc cacagtcttg 600
ggactcttcc gggtgacccc cgaaatccca gccggactcc cttcaccgag gtcggaataa 660
<210>16
<211>660
<212>DNA
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白的基因
<400>16
atgcatcatc atcatcatca cgattctaag gccgtcttcc tcgcctgccc ctgggccgtg 60
tccggggctc gcgcctcgcc cggctccgcg gccagcccga gactccgcga gggtcccgag 120
ctttcgcccg acgatcccgc cggcctcttg gacctgcggc agggcatgtt tgcgcagctg 180
gtggcccaaa atgttctgct gatcgatggg cccctgagct ggtacagtga cccaggcctg 240
gcaggcgtgt ccccgacggg gggcctgagc tacaaagagg acacgaagga gctggtggtg 300
gccaaggctg gagtctacta tgtcttcttt caactagagc tgcggcgtgt ggtggccggc 360
gagggctcag gctccgtttc acttgcgctg cacctgcagc cactgcgctc tgctgctggg 420
gccgccgccc tggctttgac cgtggacctg ccacccgcct cctccgaggc tcggaactcg 480
gccttcggtt tccagggccg cttgctgcac ctgagtgccg gccagcgcct gggcgtccat 540
cttcacactg aggccagggc acgccatgcc tggcagctta cccagggcgc cacagtcttg 600
ggactcttcc gggtgacccc cgaaatccca gccggactcc cttcaccgag gtcggaataa 660
<210>17
<211>218
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>17
Met Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg
1 5 10 15
Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
20 25 30
Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln
35 40 45
Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp
50 55 60
Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser
65 70 75
Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val
80 85 90
Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu
95 100 105
Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala
110 115 120
Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu
125 130 135
Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn
140 145 150
Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly
155 160 165
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
170 175 180
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg
185 190 195
Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
200 205 210
Leu Glu His His His His His His
215
<210>18
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>18
Met Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg
1 5 10 15
Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
20 25 30
Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln
35 40 45
Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp
50 55 60
Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser
65 70 75
Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val
80 85 90
Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu
95 100 105
Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala
110 115 120
Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu
125 130 135
Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn
140 145 150
Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly
155 160 165
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
170 175 180
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg
185 190 195
Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
200 205 210
Arg Leu Glu His His His His His His
215
<210>19
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>19
Met Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg
1 5 10 15
Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
20 25 30
Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln
35 40 45
Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp
50 55 60
Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser
65 70 75
Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val
80 85 90
Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu
95 100 105
Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala
110 115 120
Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu
125 130 135
Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn
140 145 150
Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly
155 160 165
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
170 175 180
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg
185 190 195
Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
200 205 210
Ile Leu Glu His His His His His His
215
<210>20
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>20
Met Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg
1 5 10 15
Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
20 25 30
Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln
35 40 45
Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp
50 55 60
Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser
65 70 75
Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val
80 85 90
Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu
95 100 105
Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala
110 115 120
Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu
125 130 135
Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn
140 145 150
Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly
155 160 165
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
170 175 180
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg
185 190 195
Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
200 205 210
Pro Leu Glu His His His His His His
215
<210>21
<211>216
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>21
Met His His His His His His Ala Val Phe Leu Ala Cys Pro Trp
1 5 10 15
Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser Pro
20 25 30
Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly
35 40 45
Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln
50 55 60
Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro
65 70 75
Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu
80 85 90
Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val
95 100 105
Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser
110 115 120
Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala
125 130 135
Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala
140 145 150
Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu
155 160 165
Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr
170 175 180
Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr
185 190 195
Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu
200 205 210
Pro Ser Pro Arg Ser Glu
215
<210>22
<211>217
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>22
Met His His His His His His Cys Ala Val Phe Leu Ala Cys Pro
1 5 10 15
Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser
20 25 30
Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala
35 40 45
Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala
50 55 60
Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
65 70 75
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys
80 85 90
Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr
95 100 105
Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly
110 115 120
Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser
125 130 135
Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro
140 145 150
Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
155 160 165
Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His
170 175 180
Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala
185 190 195
Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly
200 205 210
Leu Pro Ser Pro Arg Ser Glu
215
<210>23
<211>217
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>23
Met His His His His His His Met Ala Val Phe Leu Ala Cys Pro
1 5 10 15
Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser
20 25 30
Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala
35 40 45
Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala
50 55 60
Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
65 70 75
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys
80 85 90
Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr
95 100 105
Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly
110 115 120
Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser
125 130 135
Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro
140 145 150
Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
155 160 165
Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His
170 175 180
Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala
185 190 195
Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly
200 205 210
Leu Pro Ser Pro Arg Ser Glu
215
<210>24
<211>217
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>24
Met His His His His His His Tyr Ala Val Phe Leu Ala Cys Pro
1 5 10 15
Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser
20 25 30
Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala
35 40 45
Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala
50 55 60
Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
65 70 75
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys
80 85 90
Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr
95 100 105
Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly
110 115 120
Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser
125 130 135
Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro
140 145 150
Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
155 160 165
Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His
170 175 180
Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala
185 190 195
Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly
200 205 210
Leu Pro Ser Pro Arg Ser Glu
215
<210>25
<211>217
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>25
Met His His His His His His Lys Ala Val Phe Leu Ala Cys Pro
1 5 10 15
Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser
20 25 30
Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala
35 40 45
Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala
50 55 60
Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
65 70 75
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys
80 85 90
Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr
95 100 105
Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly
110 115 120
Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser
125 130 135
Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro
140 145 150
Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
155 160 165
Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His
170 175 180
Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala
185 190 195
Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly
200 205 210
Leu Pro Ser Pro Arg Ser Glu
215
<210>26
<211>218
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>26
Met His His His His His His Leu Cys Ala Val Phe Leu Ala Cys
1 5 10 15
Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala
20 25 30
Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro
35 40 45
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
50 55 60
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser
65 70 75
Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr
80 85 90
Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr
95 100 105
Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu
110 115 120
Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg
125 130 135
Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro
140 145 150
Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly
155 160 165
Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu
170 175 180
His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly
185 190 195
Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala
200 205 210
Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>27
<211>218
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>27
Met His His His His His His Ile Lys Ala Val Phe Leu Ala Cys
1 5 10 15
Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala
20 25 30
Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro
35 40 45
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
50 55 60
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser
65 70 75
Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr
80 85 90
Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr
95 100 105
Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu
110 115 120
Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg
125 130 135
Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro
140 145 150
Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly
155 160 165
Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu
170 175 180
His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly
185 190 195
Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala
200 205 210
Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>28
<211>218
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>28
Met His His His His His His Asp Lys Ala Val Phe Leu Ala Cys
1 5 10 15
Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala
20 25 30
Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro
35 40 45
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
50 55 60
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser
65 70 75
Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr
80 85 90
Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr
95 100 105
Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu
110 115 120
Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg
125 130 135
Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro
140 145 150
Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly
155 160 165
Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu
170 175 180
His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly
185 190 195
Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala
200 205 210
Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>29
<211>218
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>29
Met His His His His His His Thr Glu Ala Val Phe Leu Ala Cys
1 5 10 15
Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala
20 25 30
Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro
35 40 45
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
50 55 60
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser
65 70 75
Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr
80 85 90
Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr
95 100 105
Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu
110 115 120
Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg
125 130 135
Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro
140 145 150
Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly
155 160 165
Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu
170 175 180
His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly
185 190 195
Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala
200 205 210
Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>30
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>30
Met His His His His His His Ile Tyr Met Ala Val Phe Leu Ala
1 5 10 15
Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala
20 25 30
Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
35 40 45
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu
50 55 60
Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr
65 70 75
Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser
80 85 90
Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val
95 100 105
Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly
110 115 120
Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu
125 130 135
Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu
140 145 150
Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln
155 160 165
Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His
170 175 180
Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln
185 190 195
Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro
200 205 210
Ala Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>31
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>31
Met His His His His His His Thr Leu Val Ala Val Phe Leu Ala
1 5 10 15
Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala
20 25 30
Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
35 40 45
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu
50 55 60
Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr
65 70 75
Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser
80 85 90
Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val
95 100 105
Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly
110 115 120
Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu
125 130 135
Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu
140 145 150
Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln
155 160 165
Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His
170 175 180
Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln
185 190 195
Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro
200 205 210
Ala Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>32
<211>219
<212>PRT
<213>人工序列
<220>
<223>具有CD137L功能的重组蛋白
<400>32
Met His His His His His His Asp Ser Lys Ala Val Phe Leu Ala
1 5 10 15
Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala
20 25 30
Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
35 40 45
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu
50 55 60
Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr
65 70 75
Ser Asp Pro Gly Leu Ala Gly Val Ser Pro Thr Gly Gly Leu Ser
80 85 90
Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val
95 100 105
Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly
110 115 120
Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu
125 130 135
Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu
140 145 150
Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln
155 160 165
Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His
170 175 180
Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln
185 190 195
Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro
200 205 210
Ala Gly Leu Pro Ser Pro Arg Ser Glu
215
<210>33
<211>13
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.1-4的上游引物
<400>33
tcatatggcc gtcttcctcg cct 13
<210>34
<211>14
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.1的下游引物
<400>34
tctcgagttc cgacctcggt gaag 14
<210>35
<211>17
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.2的下游引物
<400>35
tctcgagacg ttccgacctc ggtgaag 17
<210>36
<211>17
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.3的下游引物
<400>36
tctcgaggat ttccgacctc ggtgaag 17
<210>37
<211>17
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.4的下游引物
<400>37
tctcgagcgg ttccgacctc ggtgaag 17
<210>38
<211>35
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.5-16的下游引物
<400>38
tctcgagtta ttccgacctc ggtgaaggga gtccg 35
<210>39
<211>41
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.5的上游引物
<400>39
tcatatgcat catcaccatc atcatgccgt cttcctcgcc t 41
<210>40
<211>44
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.6的上游引物
<400>40
tcatatgcat catcatcacc atcattgcgc cgtcttcctc gcct 44
<210>41
<211>44
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.7的上游引物
<400>41
tcatatgcat catcatcacc atcatatggc cgtcttcctc gcct 44
<210>42
<211>44
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.8的上游引物
<400>42
tcatatgcat catcatcacc atcattacgc cgtcttcctc gcct 44
<210>43
<211>44
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.9的上游引物
<400>43
tcatatgcat catcatcacc atcataaggc cgtcttcctc gcct 44
<210>44
<211>47
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.10的上游引物
<400>44
tcatatgcat catcatcatc accatttatg cgccgtcttc ctcgcct 47
<210>45
<211>47
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.11的上游引物
<400>45
tcatatgcat catcatcatc accatattaa ggccgtcttc ctcgcct 47
<210>46
<211>47
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.12的上游引物
<400>46
tcatatgcat catcatcatc accatgataa ggccgtcttc ctcgcct 47
<210>47
<211>47
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.13的上游引物
<400>47
tcatatgcat catcatcatc accatacgga agccgtcttc ctcgcct 47
<210>48
<211>50
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.14的上游引物
<400>48
tcatatgcat catcatcatc atcacatcta catggccgtc ttcctcgcct 50
<210>49
<211>50
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.15的上游引物
<400>49
tcatatgcat catcatcatc atcacactct tgttgccgtc ttcctcgcct 50
<210>50
<211>50
<212>DNA
<213>人工序列
<220>
<223>SEQ ID NO.16的上游引物
<400>50
tcatatgcat catcatcatc atcacgattc taaggccgtc ttcctcgcct 50
Claims (8)
1.一种编码具有CD137L功能的蛋白的基因，它选自SEQ ID NO.1至SEQ ID NO.16所示之一的核苷酸序列。
2.一种具有CD137L功能的蛋白，其特征在于该蛋白为NM_003811中第147位至第773位核苷酸表示的CD137L胞外区蛋白和His tag标签通过1-3个氨基酸的连接肽融合而成，所述1-3个氨基酸的连接肽选自下列任意一种：Leu-Glu，Arg-Leu-Glu，Ile-Leu-Glu，Pro-Leu-Glu，Cys，Met，Tyr，Lys，Leu-Cys，Ile-Lys，Asp-Lys，Thr-Glu，Ile-Tyr-Met，Thr-Leu-Val，Thr-Leu-Val和Asp-Ser-Lys。
3.根据权利要求2所述的具有CD137L功能的蛋白，其特征在于它选自SEQ IDNO.17至SEQ ID NO.32所示之一的氨基酸序列。
4.如权利要求2或3所述的具有CD137L功能的蛋白的制备方法，其特征在于包括下列步骤：
将编码权利要求2或3所述具有CD137L功能的蛋白的任一核苷酸序列插入表达载体，转化大肠杆菌，获得能够表达具有CD137L功能的蛋白的基因工程菌，通过液体培养基因工程菌，经亲和层析纯化制得具有CD137L功能的蛋白。
5.权利要求1所述的具有CD137L功能的蛋白的基因在制备调节机体免疫力的药物中的应用。
6.根据权利要求5所述应用，其特征在于调节机体免疫力的药物为调节机体T细胞增殖和机体细胞因子IL-2的合成与分泌的药物。
7.权利要求2或3所述的具有CD137L功能的蛋白在制备调节机体免疫力的药物中的应用。
8.根据权利要求7所述应用，其特征在于调节机体免疫力的药物为调节机体T细胞增殖和机体细胞因子IL-2的合成与分泌的药物。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910233712.7A CN101698852B (zh) | 2009-10-23 | 2009-10-23 | 具有cd137l功能的蛋白或多肽及其基因和应用 |
| PCT/CN2010/077978 WO2011047631A1 (zh) | 2009-10-23 | 2010-10-22 | 具有cd137l功能的蛋白或多肽及其基因和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN200910233712.7A CN101698852B (zh) | 2009-10-23 | 2009-10-23 | 具有cd137l功能的蛋白或多肽及其基因和应用 |
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| Publication Number | Publication Date |
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| CN101698852A CN101698852A (zh) | 2010-04-28 |
| CN101698852B true CN101698852B (zh) | 2014-08-13 |
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| CN101698852B (zh) * | 2009-10-23 | 2014-08-13 | 江苏先声药物研究有限公司 | 具有cd137l功能的蛋白或多肽及其基因和应用 |
| CN103232543B (zh) * | 2013-05-06 | 2014-12-03 | 中国药科大学 | 具有Tumstatin活性的重组蛋白Tumstatin-CD137L4及其制备和应用 |
| CN103214584B (zh) * | 2013-05-06 | 2014-07-09 | 中国药科大学 | 具有抑制肿瘤微环境血管再生和激活适应性免疫应答双功能的融合蛋白及其基因和应用 |
| CN105906719B (zh) * | 2016-05-09 | 2019-06-11 | 扬州大学 | 一种自聚肽融合cd151蛋白及其制备方法与应用 |
| CN113842456B (zh) * | 2020-06-28 | 2022-07-26 | 上海齐鲁制药研究中心有限公司 | 一种抗人4-1bb的单克隆抗体制剂及其用途 |
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| CA2515100A1 (en) * | 2003-02-06 | 2004-08-19 | Micromet Ag | Trimeric polypeptide construct to induce an enduring t cell response |
| ES2567704T3 (es) * | 2007-07-10 | 2016-04-26 | Apogenix Gmbh | Proteínas de fusión de colectina de la superfamilia de TNF |
| CN101698852B (zh) * | 2009-10-23 | 2014-08-13 | 江苏先声药物研究有限公司 | 具有cd137l功能的蛋白或多肽及其基因和应用 |
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Non-Patent Citations (4)
| Title |
|---|
| Analysis of CD137 and CD137L Expression in Human Primary Tumor Tissues;wang qun,et al.;《Croat Med J.》;20080430;第49卷(第2期);192-200 * |
| wang qun,et al..Analysis of CD137 and CD137L Expression in Human Primary Tumor Tissues.《Croat Med J.》.2008,第49卷(第2期),192-200. |
| 噬菌体展示技术在鼠CD137L活性研究中的应用;夏西燕等;《山东医药》;20061231;第46卷(第2期);4-6 * |
| 夏西燕等.噬菌体展示技术在鼠CD137L活性研究中的应用.《山东医药》.2006,第46卷(第2期),4-6. |
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| Publication number | Publication date |
|---|---|
| WO2011047631A1 (zh) | 2011-04-28 |
| CN101698852A (zh) | 2010-04-28 |
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