CN111087473B - 一种SIRPa-Fc-IL21融合蛋白及其应用 - Google Patents
一种SIRPa-Fc-IL21融合蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了一种SIRPa‑Fc‑IL21融合蛋白及其应用。一种SIRPa‑Fc‑IL21融合蛋白，氨基酸序列如SEQ ID NO.1所示。本发明所述的融合蛋白在制备治疗肿瘤的药物中的应用；优选在制备治疗淋巴癌的药物中的应用。本发明构建了人SIRPa‑Fc‑IL21融合蛋白，通过SIRPa和肿瘤细胞表面配体CD47结合阻断CD47与免疫细胞表面SIRPα之间的相互作用，同时利用IL21刺激免疫细胞增殖的作用，增加体内免疫细胞的数量，加强免疫细胞的杀伤作用。
Description
技术领域
本发明属于生物医药领域，涉及一种SIRPa-Fc-IL21融合蛋白及其应用。
背景技术
CD47也被称为整合素相关蛋白(integrin associated protein，IAP)。CD47广泛的表达于细胞的表面，可与信号调节蛋白α(Signal regulatory protein α,SIRP α)、血小板反应蛋白(thrombospondin-1,TSP1)以及整合素(integrins)相互作用，介导细胞凋亡、增殖、免疫等一系列反应。
Oldenborg等人证实CD47是细胞表面一个重要的"self"标记，是调节巨噬细胞吞噬作用的一个重要信号。CD47可以与巨噬细胞表面SIRPα结合，磷酸化其ITIM，随后招募SHP-1蛋白，产生一系列的级联反应抑制巨噬细胞的吞噬作用。肿瘤细胞高表达CD47躲避吞噬肿瘤细胞有一系列躲避人体免疫系统追杀的方案，包括分泌免疫抑制因子、下调MHC I表达，以及上调PD-L1抑制CD8+T细胞活性。聪明的肿瘤细胞当然不会放过CD47这个完美的掩体。不同的研究表明，几乎所有的肿瘤细胞和组织都高表达CD47，是对应正常细胞和组织的3倍。通过CD47这个"self"信号，肿瘤细胞有效的躲避了巨噬细胞的吞噬作用。
SIRPα是CD47的受体，SIRPa蛋白与肿瘤微环境中的CD47结合，通过竞争性结合来降低T细胞表面的SIRPa与肿瘤细胞表面配体CD47的结合进而阻断信号通路的持续激活，从而充分发挥融合蛋白清除免疫抑制作用。阻断CD47与巨噬细胞SIRPα之间的相互作用可增强巨噬细胞和中性粒细胞的吞噬作用进而消灭肿瘤细胞。
IL21的生物学功能主要是刺激T细胞增殖、NK细胞增殖和分化以及CD40特异性应答B细胞的增殖。IL21可以有效的增加T细胞的增殖增强肿瘤免疫治疗作用。
抗体的Fc端，具有高稳定性，长效性，能够有效保证融合蛋白的活性同时增加融合蛋白的效用。
目前尚未见SIRPa-Fc与IL21融合蛋白的相关报道。
发明内容
本发明的目的是提供一种较之SIRPa-Fc具有更强肿瘤抑制作用的SIRPa-Fc与IL21融合蛋白。
本发明的另一目的是提供该融合蛋白的编码基因。
本发明的又一目的是提供该融合蛋白的应用。
本发明的目的可通过以下技术方案实现：
一种SIRPa-Fc-IL21融合蛋白，氨基酸序列如SEQ ID NO.1所示。本发明融合片段中SIRPa片段来自人SIRPa的胞外段区域，氨基酸序列如SEQ ID NO.2所示；Fc来自人IgG的Fc片段，氨基酸序列如SEQ ID NO.3所示，IL21片段来自人IL21的胞外段区域，氨基酸序列如SEQ ID NO.4所示；所述的SIRPa-Fc-IL21融合蛋白依次由所述的SIRPa片段、Fc片段、接头氨基酸(G4S)3和IL21片段串联而成。
本发明所述的融合蛋白的编码基因；优选核苷酸序列如SEQ ID NO.5所示。
含有本发明所述编码基因的重组表达载体。
所述的重组表达载体，优选将所述的编码基因克隆到pCDNA3.4质粒NotI/XbaI酶切位点间所得。
本发明所述的融合蛋白在制备治疗肿瘤的药物中的应用；优选在制备治疗淋巴癌的药物中的应用。
本发明所述的编码基因、所述的重组表达载体在制备治疗肿瘤的药物中的应用；优选在制备治疗淋巴癌的药物中的应用。
一种治疗淋巴癌的药物，包含本发明所述的融合蛋白。
有益效果：
本发明构建了人SIRPa-Fc-IL21融合蛋白，通过SIRPa和肿瘤细胞表面配体CD47结合阻断CD47与免疫细胞表面SIRPα之间的相互作用，同时利用IL21刺激免疫细胞增殖的作用，增加体内免疫细胞的数量，加强免疫细胞的杀伤作用。动物实验结果显示，本发明SIRPa-Fc-IL21融合蛋白中SIRPa、Fc及IL21三个片段在功能上发挥了协同作用，SIRPa-Fc-IL21融合蛋白较之单独使用SIRPa-Fc片段和单独使用IL21在抑瘤效果方面具有显著提高。
附图说明
图1载体图谱
A表达载体pCDNA3.4的图谱,B克隆载体PUC57的图谱，C重组质粒图谱
图2PCR产物电泳分离图
泳道1-2：A片段，泳道3-4：B片段，泳道：C片段，泳道7-8：D片段
图3.阳性克隆筛选鉴定
图4.质粒酶切鉴定
图5.ELISA检测曲线
图6SDS-PAGE检测图
M：Marker，Lane1：hSIRPa-hFc-hIL21还原样品,Lane2：hSIRPa-hFc-hIL21非还原样品
图7SEC-HPLC检测图
图8Binding ELISA检测结果
具体实施方式
实施例1
实验材料：引物，pCDNA3.4，PUC57载体质粒,高保真PCR聚合酶,重组酶,胶回收试剂盒，dNTP，限制性内切酶。
实验方法：
1全基因合成：
2获取四段克隆目标序列
1)A片段：引物1-8；
2)B片段：引物7-16；
3)C片段：引物15-24；
4)D片段：引物23-30，
四个片段引物1-8，7-16，15-24，23-30分别混Mix，第一轮PCR(以A片段为例)：
反应50ul体系为：
PCR反应条件：
循环25次；
72℃充分延伸3min。
第二轮PCR(以A片段为例)，反应50ul体系为：
反应条件：
循环25次；
72℃充分延伸5min。
PCR反应后，使用1.8％的琼脂糖胶对PCR产物进行电泳分离，在紫外透射仪中用手术刀切下含有目的PCR产物的琼脂糖凝胶，图2显示PCR产物的电泳图。1、2为第一个克隆目标片段A(628bp)，3、4为第一个克隆目标片段B(790bp)，5、6为第一个克隆目标片段C(802bp)，7、8为第一个克隆目标片段D(628bp)。胶回收上述步骤克隆目标片段PCR产物(参考Axygen产品说明书)准备连接实验。
3载体酶切线性：
| PUC57 | 6ul(1.5ug) |
| 10×buffer | 10μl |
| EcoRV | 2μl |
| ddH20 | 32μl |
| 共计 | 50μl |
| pCDNA3.4 | 6ul(1.5ug) |
| 10×buffer | 10μl |
| NotI | 2μl |
| XbaI | 2μl |
| ddH20 | 30μl |
| 共计 | 50μl |
根据表达中的酶切体系，载体质粒用对应的限制性酶切。将上述体系置37℃水浴2h，然后65℃，15min灭活限制性内切酶。酶切产物胶回收。
4 PUC57克隆并测序：
将4段克隆目标片段克隆至PUC57载体里面，获得测序正确的质粒A，质粒B，质粒C，质粒D。
5 pCNDA3.4克隆并测序
用1号和8号引物扩质粒A，用7号和16号引物扩质粒B，用15号和24号引物扩质粒C，用25号和30号引物扩质粒D，获得4个PCR产物用琼脂糖电泳胶回收后，用1号和30号引物扩4个PCR片段，拼全长获得克隆片段，将正确的全长片段琼脂糖电泳胶回收后，重组至pCDNA3.4载体中。
6菌落筛选实验
1)挑取过夜平板中单菌落。
2)用BI-seqF，BI-seqR进行菌落PCR(图3)。
3)所得产物以1.2％琼脂糖电泳鉴定阳性克隆
4)随机选择4个阳性菌，阳性菌液用下面的引物测序。
BI-seqF 5'-GATCGCCTGGAGACGCCATC-3'(SEQ ID NO.36)
BI-seqR 5'-AGCGTAAAAGGAGCAACATAGT-3'(SEQ ID NO.37)
seqF1 5'-CCACACCTCAGCACACAGTG-3'(SEQ ID NO.38)
seqF2 5'-GACCTGAAGGTCTCAGCCCA-3'(SEQ ID NO.39)
seqF3 5'-AGTACAAGTGCAAGGTCTCC-3'(SEQ ID NO.40)
5)选择测序正确克隆的阳性菌液，扩大培养后抽提低内毒素质粒，测序验证。
实验结论：图4泳道3所示，用NotI和XbaI双酶切鉴定分析，切下来的片段大小约2300bp，与片段大小一致，这些结果表明SIRPA-FC-IL21重组质粒构建成功。阳性克隆寄送南京擎科生物科技有限公司进行测序，测序结果与目标序列一致，进一步说明重组载体构建成功。
实施例2蛋白表达
实验名称：细胞培养与表达
实验材料：摇床、离心机、水浴锅、293 CD05 Medium培养液、转染试剂、各种规格的移液管、各种规格的摇瓶。
实验方法：
1细胞培养：HEK293细胞
| 步骤 | 培养条件 | 传代前密度 | 传代后密度及体积 |
| 1.细胞复苏 | 120rpm，8％CO2，37℃培养 | 0 | 0.25×10<sup>6</sup>个/ml |
| 2.细胞初次传代 | 120rpm，8％CO2，37℃培养 | <u>1.1×10<sup>6</sup>个/ml</u> | 0.5×10<sup>6</sup>个/ml |
| 3.细胞再次传代 | 120rpm，8％CO2，37℃培养 | <u>2.4×10<sup>6</sup>个/ml</u> | 0.4×10<sup>6</sup>个/ml |
| 4.瞬转 | 120rpm，8％CO2，37℃培养 | <u>2.7×10<sup>6</sup>个/ml</u> | 1.5×10<sup>6</sup>/ml |
2瞬时转染与表达
溶液1：用培养液稀释质粒，混匀
溶液2：用培养液稀释转染试剂，混匀
将溶液2加入溶液1中，混匀，37℃孵育15分钟后，将混合转染液逐滴加入细胞液中，边摇边加，放至摇床培养。
培养表达一周，收集上清，8000rpm离心5min。
3 ELISA检测预测上清表达量
取离心后的上清300uL(首孔取原液，后续1∶5稀释)，进行ELISA检测。
实验结果：
选择吸光度在0.6-1.2范围内的数值带入公式(图5)
计算可知：该蛋白预测表达量为2.6mg/L。
实施例3
1细胞培养上清纯化
实验材料：蠕动泵、搅拌器、玻璃纯化柱、1XPBS、柠檬酸钠、Protein At Beads4FF、各种规格的离心管
实验方法：Protein A亲和层析柱纯化
1)装柱：填料混匀后，用移液枪吸取填料悬浮液加入层析柱中。将泵，连接管路，层析柱三者连接好，做好柱子标签。
2)用1XPBS溶液平衡层析柱。
3)平衡后上样细胞培养上清。
4)上样结束后，先用1XPBS溶液洗杂。
5)洗杂结束后，1XPBS溶液走净，用柠檬酸钠溶液(PH3.4)洗脱，分管收集，每管约500ul。共收集5管，使用NanoDrop仪器读取280nm吸光度值。
6)混合蛋白：将有浓度的蛋白混合到适宜的离心管中，进行透析前体积浓度记录。
7)蛋白透析：将混合蛋白吸至透析袋中，扎紧透析袋，做好标记，放至含1XPBS溶液的1L烧杯中置于搅拌器上帮助透析。
8)透析结束后，用一次性无菌注射器将蛋白取出并取样，交于质控。
2纯度检测
A.SDS-PAGE检测
实验材料：电泳仪、垂直电泳槽、恒温金属浴，30％丙烯酰胺，1.5M Tris-HCl，1MTris-HCl，10％SDS，2×SDS-PAGELoading Buffer，10％AP，5×SDS-PAGE电泳缓冲液，脱色液，染色液实验方法：
1)样品处理
还原样品处理：还原上样缓冲液中入3.0ug蛋白，99℃处理10分钟。
非还原样品处理：非还原上样缓冲液中入3.0ug蛋白。
2)电泳
先用80V电压跑15min，再用170V电压跑40min。
3)凝胶的染色及脱色
将凝胶放入染色液中，沸水中煮15min后置60rpm摇床摇晃1h。换脱色液在沸水中煮15min后置60rpm摇床摇晃1h，更换脱色液，置60rpm摇床继续摇晃2h，拍照保存。
实验结果：见图6。由图可知：该蛋白理论分子量为83KDa，实际分子量约为85-100Kda，纯度＞90％。
3 SEC-HPLC检测
实验仪器及材料：高效液相色谱仪、凝胶色谱柱、去离子水、流动相(Na2HPO4.12H2O、NaH2PO4. 2H2O、NaCl)
实验方法：
使用高效液相色谱仪LC-20AT及凝胶色谱柱进行SEC实验，实验条件如下：
| 参数 | 数值 |
| 流速 | 1ml/min |
| 样品浓度 | <u>0.58</u>mg/ml |
| 进样量 | 20μl |
| 柱温 | 35℃ |
| 检测波长 | 214nm，280nm |
| 采集时间 | 15min |
将水更换为流动相，流速缓慢升至1.000ml/min，至基线平稳。取50ul蛋白至对应编号的进样瓶中，将样品瓶放入仪器相应的位置，进样时间是15min，分析处理数据并保存，将流动相更换成去离子水，冲洗1.5h。
实验结果：见图7。
由图可知：214nm纯度是89.3％，280nm纯度是80％。
4内毒素检测
实验仪器及材料：旋涡振荡器、电热恒温培养箱、内毒素工作标准品、鲎试剂、内毒素检查用水(安度斯)
实验方法：
1)样品阳性对照液配制：2倍供试液浓度溶液+内毒素标准品(0.5EU/ml)，1∶1混合
2)供试液制备：
供试液制备：样品稀释倍数：MVD＝C·L/λ＝(0.58mg/ml*5EU/mg)÷0.25EU/ml＝ 11.6
*注：MVD：供试品最大有效稀释倍数
L：供试品细菌内毒素限值(5EU/mg)
C：供试品浓度
λ：鲎试剂标示灵敏度(0.25EU/ml)
110ul供试液取样量＝0.58mg/ml÷11.6*110ul＝5.5ug
封闭管口，轻轻摇匀，垂直放入37℃恒温培养箱孵育60min
实验结果：鲎试剂状态是澄清透明不凝固。
实验结论：通过内毒素检测，结果是<5EU/mg。
实施例4 Binding ELISA检测
实验验证：人SIRPa-Fc-IL21融合蛋白与人CD47/mFc和IL21R/hFc有无结合。
1检测器材及试剂
酶标仪：VersaMax，洗板机：ELX405R，单道、多道移液枪，加样槽
包被缓冲液、PBS/吐温、1X PBS缓冲液、3％BSA溶液、TMB溶液、1M盐酸溶液
Human SIRPa-hFc-IL21、Human CD47/hFc、Human IL21R/hFc
2 Human CD47/hFc、Human IL21R/hFc蛋白生物素标记
取3ul配制好的生物素酯溶液加至300ul Fc融合蛋白溶液(0.5mg/mL)中室温反应1小时。加入3.3ul 0.5M Tris(pH＝8)室温放置1小时以终止反应。1X PBS溶液过夜透析。按融合蛋白的储存方法保存标记蛋白。蛋白生物素标记后标为Human CD47/HFc-Biotin、Human IL21R/hFc-Biotin。
3检测过程
3.1.包被
(1)根据实验所需量包被酶标板，将蛋白Human SIRPa-hFc-IL21
浓度稀释为20ug/ml，混匀后倒入加样槽。用30-300ul多道移液枪向酶标板各孔中加入50ul稀释后的抗体，轻轻振荡酶标板使抗体覆盖酶标板孔底。
(2)封口膜封板，4℃过夜孵育。
3.2封闭
(1)将4℃过夜孵育后的酶标板置于洗板机上，用1XPBS缓冲液洗涤3次后，加入200ul封闭液(3％BSA)进行封闭。
(2)加入封闭液后在培养箱中37℃孵育60min。
(3)将酶标板放于洗板机上，用PBS/吐温缓冲液洗涤5次(可以将酶标板内的液体甩干后再冲洗，以防冲洗过程中堵塞管道。)
3.3加样
(1)将样品蛋白Human CD47/HFc-Biotin浓度稀释至0.1mg/ml，以0.1ug/ml为起始浓度，1/2梯度稀释；Human IL21R/hFc-Biotin浓度稀释至0.1mg/ml，以0.25ug/ml为起始浓度，1/2梯度稀释。
(2)封口膜封板后在37℃作用90min。
(3)将酶标板放于洗板机上，用PBS/吐温缓冲液洗涤5次。
3.4加A-HRP：
加入稀释的辣根过氧化物酶标记的亲和素(A-HRP)，封口膜封板后室温(22℃)反应30min，用PBS/吐温缓冲液冲洗5次。
3.5底物显色：
(1)于上述各反应孔中加入TMB 0.1ml，于37℃反应30分钟。再加1M HCl 0.1ml以终止反应。
(2)酶标仪450nm处读取光密度值。
4.实验数据：
4.1包被SIRPa-hFc-IL21，检测CD47/hFc(图8A),EC50＝12.51ng/mL
| CD47/hFc onc.(ng/mL) | OD450 | |
| A | 100 | 2.492 |
| B | 50 | 2.50 |
| C | 25 | 2.123 |
| D | 12.5 | 1.361 |
| E | 6.25 | 0.499 |
| F | 3.125 | 0.236 |
| G | 0 | 0.097 |
4.2包被SIRPa-hFc-IL21，检测IL21R/hFc(图8B),EC50＝28.49ng/mL
实施例5动物实验
实验验证：鼠SIRPa-Fc-IL21融合蛋白、SIRPa-Fc融合蛋白与IL21-Fc融合蛋白对人淋巴瘤模型小鼠的肿瘤治疗效果。
1.检测器材及试剂
鼠淋巴瘤模型小鼠28只，SIRPa-Fc-IL21融合蛋白、SIRPa-Fc融合蛋白、IL21-Fc融合蛋白、PBS、注射器、解剖剪、纱布、电子天平等。
2.实验方法
2.1淋巴瘤模型小鼠购买28只(已培养3周)，随机为4组
2.2a组注射200ul PBS；b组注射SIRPa-Fc-IL21融合蛋白200ug(200ul PBS溶解)；c组注射SIRPa-Fc融合蛋白200ug(200ul PBS溶解)；d组注射IL21-Fc融合蛋白200ug(200ulPBS溶解)。每天注射，持续一周。
2.3切除肿瘤进行称重，分析结果。
3.实验结果
| 组 | a | b | c | d |
| 肿瘤重量(平均) | 1.34 | 0.35 | 0.56 | 0.93 |
由实验结果可见，SIRPa-Fc-IL21融合蛋白对淋巴瘤细胞具有显著的杀伤作用。
序列表
<110> 泰州市百英生物科技有限公司
<120> 一种SIRPa-Fc-IL21融合蛋白及其应用
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Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
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Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
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Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly
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Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly
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Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr
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Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
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Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
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Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
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Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
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Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
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Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
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Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
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Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
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Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
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Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
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Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
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Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
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Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
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Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His
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Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn
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Glu Arg Asn Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
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Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
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Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
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Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
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Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
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Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
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Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
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Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
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Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
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Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
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Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Arg His Met Ile
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Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val
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Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr
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Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys
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Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys
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Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
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Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
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Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
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Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly
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Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly
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Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr
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Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
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Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
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Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
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Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
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Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
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Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
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Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
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Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
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Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
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Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
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Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
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Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
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Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
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Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
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Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn
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Glu Arg Asn
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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
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Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
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Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
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Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
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Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
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Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
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Glu Asp Ser
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<210> 20
<211> 2333
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
agaagacacc gggaccgatc cagcctccgg acgcggccgc caccatggag cccgccggcc 60
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caggagtggc gggtgaggag gagctgcagg tgattcagcc tgacaagtcc gtgttggttg 180
cagctggaga gacagccact ctgcgctgca ctgcgacctc tctgatccct gtggggccca 240
tccagtggtt cagaggagct ggaccaggcc gggaattaat ctacaatcaa aaagaaggcc 300
acttcccccg ggtaacaact gtttcagacc tcacaaagag aaacaacatg gacttttcca 360
tccgcatcgg taacatcacc ccagcagatg ccggcaccta ctactgtgtg aagttccgga 420
aagggagccc cgatgacgtg gagtttaagt ctggagcagg cactgagctg tctgtgcgtg 480
ccaaaccctc tgcccccgtg gtatcgggcc ctgcggcgag ggccacacct cagcacacag 540
tgagcttcac ctgcgagtcc cacggcttct cacccagaga catcaccctg aaatggttca 600
aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccgtagga gagagcgtgt 660
cctacagcat ccacagcaca gccaaggtgg tgctgacccg cgaggacgtt cactctcaag 720
tcatctgcga ggtggcccac gtcaccttgc agggggaccc tcttcgtggg actgccaact 780
tgtctgagac catccgagtt ccacccacct tggaggttac tcaacagccc gtgagggcag 840
agaaccaggt gaatgtcacc tgccaggtga ggaagttcta cccccagaga ctacagctga 900
cctggttgga gaatggaaac gtgtcccgga cagaaacggc ctcaaccgtt acagagaaca 960
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acctgaaggt ctcagcccac ccgaaggagc agggctcaaa taccgccgct gagaacactg 1140
gatctaatga acggaacgag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc 1200
cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca 1260
ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag 1320
accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa 1380
agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc 1440
accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag 1500
cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca 1560
ccctgccccc atcccgggag gagatgacca agaaccaggt cagcctgacc tgcctggtca 1620
aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca 1680
actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc 1740
tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg 1800
aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt aaaggaggag 1860
gaggaagcgg aggaggaggg agcggaggag ggggaagcca agatcgccac atgattagaa 1920
tgcgtcaact tatagatatt gttgatcagc tgaaaaatta tgtgaatgac ttggtccctg 1980
aatttctgcc agctccagaa gatgtagaga caaactgtga gtggtcagct ttttcctgct 2040
ttcagaaggc ccaactaaag tcagcaaata caggaaacaa tgaaaggata atcaatgtat 2100
caattaaaaa gctgaagagg aaaccacctt ccacaaatgc agggagaaga cagaaacaca 2160
gactaacatg cccttcatgt gattcttatg agaaaaaacc acccaaagaa ttcctagaaa 2220
gattcaaatc acttctccaa aagatgattc atcagcatct gtcctctaga acacacggaa 2280
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<210> 1
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agaagacacc gggaccgatc cagcctccgg acgcggccgc caccatggag cccgccggcc 60
cggcccccgg ccgcctcggg ccgctg 86
<210> 2
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgaatcacct gcagctcctc ctcacccgcc actcctgacc aggcgcagga cgcggcgagc 60
agcaggcaga gcagcggccc gaggcg 86
<210> 3
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctgcaggtg attcagcctg acaagtccgt gttggttgca gctggagaga cagccactct 60
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<210> 4
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgttacccgg gggaagtggc cttctttttg attgtagatt aattcccggc ctggtccagc 60
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<210> 5
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttcccccggg taacaactgt ttcagacctc acaaagagaa acaacatgga cttttccatc 60
cgcatcggta acatcacccc agcagatgcc ggcac 95
<210> 6
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcacagacag ctcagtgcct gctccagact taaactccac gtcatcgggg ctccctttcc 60
ggaacttcac acagtagtag gtgccggcat ctgct 95
<210> 7
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgagctgtc tgtgcgtgcc aaaccctctg cccccgtggt atcgggccct gcggcgaggg 60
ccacacctca gcacacagtg agcttc 86
<210> 8
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tggaagtctg agagctcatt cccatttttg aaccatttca gggtgatgtc tctgggtgag 60
aagccgtggg actcgcaggt gaagctcact gtgtg 95
<210> 9
<211> 88
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gctctcagac ttccagacca acgtggaccc cgtaggagag agcgtgtcct acagcatcca 60
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<210> 10
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtcccacgaa gagggtcccc ctgcaaggtg acgtgggcca cctcgcagat gacttgagag 60
tgaacgtcct cgcgggtcag caccac 86
<210> 11
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccctcttcgt gggactgcca acttgtctga gaccatccga gttccaccca ccttggaggt 60
tactcaacag cccgtgaggg cagagaacca ggtga 95
<210> 12
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgtccgggac acgtttccat tctccaacca ggtcagctgt agtctctggg ggtagaactt 60
cctcacctgg caggtgacat tcacctggtt ctctg 95
<210> 13
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aacgtgtccc ggacagaaac ggcctcaacc gttacagaga acaaggatgg tacctacaac 60
tggatgagct ggctcctggt gaatgtatct gccca 95
<210> 14
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agaccttcag gtcatggctt ttgctgaccg ctggctgccc gtcatgctcc acctggcagg 60
tgagcttcac atcatccctg tgggcagata cattc 95
<210> 15
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgacctgaa ggtctcagcc cacccgaagg agcagggctc aaataccgcc gctgagaaca 60
ctggatctaa tgaacggaac gagcccaaat cttgt 95
<210> 21
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggttttgggg ggaagaggaa gactgacggt ccccccagga gttcaggtgc tgggcacggt 60
gggcatgtgt gagttttgtc acaagatttg ggctc 95
<210> 22
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag gtcacatgcg 60
tggtggtgga cgtgagccac gaagaccctg aggtc 95
<210> 23
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tacgtgctgt tgtactgctc ctcccgcggc tttgtcttgg cattatgcac ctccacgccg 60
tccacgtacc agttgaactt gacctcaggg tcttc 95
<210> 24
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 60
tggcaaggag tacaagtgca aggtctccaa caaag 95
<210> 25
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgggggcagg gtgtacacct gtggttctcg gggctgccct ttggctttgg agatggtttt 60
ctcgatgggg gctgggaggg ctttgttgga gacct 95
<210> 26
<211> 93
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 60
gtcaaaggct tctatcccag cgacatcgcc gtg 93
<210> 27
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gagccgtcgg agtccagcac gggaggcgtg gtcttgtagt tgttctccgg ctgcccattg 60
ctctcccact ccacggcgat gtcgct 86
<210> 28
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca 60
gcaggggaac gtcttctcat gctccgtgat gcatg 95
<210> 29
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ccctcctcct ccgcttcctc ctcctccttt acccggagac agggagaggc tcttctgcgt 60
gtagtggttg tgcagagcct catgcatcac ggagc 95
<210> 30
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agcggaggag gagggagcgg aggaggggga agccaagatc gccacatgat tagaatgcgt 60
caacttatag atattgttga tcagctgaaa aatta 95
<210> 31
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agcaggaaaa agctgaccac tcacagtttg tctctacatc ttctggagct ggcagaaatt 60
cagggaccaa gtcattcaca taatttttca gctga 95
<210> 32
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
cagctttttc ctgctttcag aaggcccaac taaagtcagc aaatacagga aacaatgaaa 60
ggataatcaa tgtatcaatt aaaaag 86
<210> 33
<211> 95
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
tcataagaat cacatgaagg gcatgttagt ctgtgtttct gtcttctccc tgcatttgtg 60
gaaggtggtt tcctcttcag ctttttaatt gatac 95
<210> 34
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
atgtgattct tatgagaaaa aaccacccaa agaattccta gaaagattca aatcacttct 60
ccaaaagatg attcatcagc atctgt 86
<210> 35
<211> 89
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
caaattttgt aatccagagg ttgattgtcg actctagact caggaatctt cacttccgtg 60
tgttctagag gacagatgct gatgaatca 89
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gatcgcctgg agacgccatc 20
<210> 37
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
agcgtaaaag gagcaacata gt 22
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ccacacctca gcacacagtg 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gacctgaagg tctcagccca 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
agtacaagtg caaggtctcc 20
Claims (8)
1.一种SIRPa-Fc-IL21融合蛋白，其特征在于氨基酸序列如SEQ ID NO.16所示。
2.权利要求1所述的融合蛋白的编码基因。
3.根据权利要求2所述的编码基因，其特征在于核苷酸序列如SEQ ID NO.20所示。
4.含有权利要求2或3所述编码基因的重组表达载体。
5.根据权利要求4所述的重组表达载体，其特征在于是将权利要求2或3所述的编码基因克隆到pCDNA3.4质粒NotI/XbaI酶切位点间所得。
6.权利要求1所述的融合蛋白在制备治疗淋巴癌的药物中的应用。
7.权利要求2或3所述的编码基因、权利要求4或5所述的重组表达载体在制备治疗淋巴癌的药物中的应用。
8.一种治疗淋巴癌的药物，其特征在于包含权利要求1所述的融合蛋白。
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| CN102775501A (zh) * | 2012-08-03 | 2012-11-14 | 中国科学院生物物理研究所 | 用于肿瘤治疗的白细胞介素21(il-21)药物 |
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