CN112552391B - 一种重组白介素-15类似物 - Google Patents
一种重组白介素-15类似物 Download PDFInfo
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- CN112552391B CN112552391B CN202011012457.6A CN202011012457A CN112552391B CN 112552391 B CN112552391 B CN 112552391B CN 202011012457 A CN202011012457 A CN 202011012457A CN 112552391 B CN112552391 B CN 112552391B
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Abstract
本发明公开了一种IL‑15类似物，IL‑15类似物的氨基酸序列包括IL‑15氨基酸序列以及在IL‑15氨基酸序列的C末端添加的一个或多个氨基酸，该一个或多个氨基酸包括带正电的氨基酸。本发明的IL‑15类似物在大肠杆菌中高表达，表达量约为野生型IL‑15的20倍，并且其体外细胞活性无明显差异。本发明还公开了一种IL‑15类似物的偶联物，通过偶联脂肪酸链，提高了IL‑15类似物的半衰期，提高其长效性。本发明为IL‑15蛋白药物产业化奠定了基础。
Description
技术领域
本发明属于分子生物学领域，具体涉及一种重组白介素-15类似物及其表达。
背景技术
白细胞介素-15(IL-15)是一种约为12-14kD的细胞因子，天然人白介素15成熟肽含有114个氨基酸，包括4个半胱氨酸残基，Cys35与Cys85、Cys42与Cys88连接，形成的两对分子内二硫键对于保持IL-15的空间构象和生物学活性起重要作用。
与大多数细胞因子相似，IL-15可在机体正常的免疫应答中发挥作用，如促进T细胞、B细胞、自然杀伤(NK)细胞的发育等，IL-15与IL-2拥有共有的IL-2Rβ链和γ链受体，所以其生物学活性很相似。但是由于拥有不同α链受体，导致IL-2可以活化Treg细胞，以及诱导Teff和NK细胞凋亡(AICD)，临床应用受到了极大限制。而同家族的IL-15却没有Treg活化和AICD的功能，因此IL-15具有巨大的治疗潜力。
IL-15受体的α亚基与IL-15有很高的亲和力，生理状态下IL-15大多与α亚基形成复合物(IL-15-Rα)，并增强IL-15对受体的β链和γ链亚基的亲和力，激活T细胞和NK细胞。因此，ALTOR和恒瑞等公司利用α亚基的特点，用融合或非融合的方法将IL-15与α亚基(或其部分)形成复合物，并在动物试验中显示了良好的生物效价及稳定性。
IL-15受体的α亚基表达在髓系细胞表面，包括巨噬细胞，抗原提呈细胞，以及NK细胞和T细胞，同样在体内抗肿瘤中发挥着重要作用。因此IL-15-Rα复合物不依赖于体内的α亚基，虽然提高了IL-15单体的稳定性，但与天然的IL-15在功能上有着微妙的区别，这种区别可能在体内的抗肿瘤中彰显出本质的不同。
尽管，IL-15较IL-2有着更高的安全性及活性，但作为蛋白类药物，天然野生型的IL-15存在明显的药物开发瓶颈，包括原核生物和真核生物表达的表达量低、纯化困难、半衰期短等，从而难以进行产业化，所以有必要对其外源表达进行研究以获得高生产效能。此外还可在经过体外复性获取的IL-15类似物的N端或C端进行偶联人血清白蛋白结合物脂肪酸链，达到可以长效的效果，对肿瘤等治疗具有一定的意义。
发明内容
有鉴于现有技术中IL-15的生产效能低，本发明提供了一种IL-15类似物，其表达量高，纯化相对简单。另外，本发明还提供了一种IL-15类似物的偶联物，以提高IL-15的半衰期，提高其长效性。
本发明的一个方面提供了一种IL-15类似物，在一个具体实施方式中，该IL-15类似物的氨基酸序列包括IL-15氨基酸序列以及在IL-15氨基酸序列的C末端添加的一个或多个氨基酸。
进一步地，前述一个或多个氨基酸包括带正电的氨基酸。
进一步地，IL-15类似物的氨基酸序列具有如下特征：IL-15-Xa-Yb-Zc，其中，X、Y和Z代表C末端添加的氨基酸序列，a、b和c代表氨基酸个数；
X和Z为任意氨基酸或其组合，其中，a和c为0～20；Y为带正电的氨基酸或其组合、或带正电的氨基酸和其他任意氨基酸的组合，其中b为1～7。
进一步地，带正电的氨基酸为H、R和K。
可选地，前述X包括V、I、P、L、E、A、S、C、T、G中的任一个或其组合。
可选地，IL-15类似物的氨基酸序列具有如下特征：IL-15-Linker-Xa-Yb-Zc，其中，Linker代表在IL-15氨基酸序列和C末端添加的氨基酸序列之间的连接序列。
进一步地，Linker为(GGGGS)n、(GS)n或(GAPQ)n，n为0～10。
进一步地，Xa包括LPBTG，其中B为任意氨基酸；Linker为(GS)n。
可选地，IL-15类似物的氨基酸序列如下所示：IL-15-GSGSGS-HHHHHH、IL-15-GS-HHHHHH、IL-15-PLASTKKR、IL-15-LPKSAKKK、IL-15-KKKKKKK、IL-15-(GAPQGAPQ)-LVESAHHH、IL-15-GS-LVSSAHHK、IL-15-GS-LIEHHRRK、IL-15-GS-IVEHRKKK、IL-15-GS-VPKTGRRR、IL-15-GS-LVASGKK、IL-15-GS-HRKSGHHH、IL-15-GS-LPKTGRHK、IL-15-KKKTGRRH、IL-15-LPRSGRHK、IL-15-LVETHHHH、IL-15-VRPETHHH、IL-15-KKK、IL-15-RHHHH、IL-15-KRETHHHH、IL-15-GS-LPETG-GSGGSHHHHHH、IL-15-HLETGKKK、IL-15-HVESGRRR、IL-15-RRHTGKKK、IL-15-HVKTGHHH、IL-15-HVKSGRHH、IL-15-HVKSSHRH、IL-15-GSGSGSGSGS-LVKSGHHH、IL-15-RPKSGHHK、IL-15-KKC、IL-15-LHKAGKHH、IL-15-K、IL-15-KK。
进一步地，IL-15的氨基酸序列选自：
1)如SEQ ID NO.1所示氨基酸序列；
2)与SEQ ID NO.1所示的氨基酸序列通过一个或多个氨基酸取代、缺失或添加衍生产生的氨基酸序列；
3)与SEQ ID NO.1具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列。
本发明的第二个方面提供了一种核苷酸序列，其编码如上所述的IL-15类似物。
本发明的第三个方面提供了一种IL-15类似物的制备方法，在原核系统中表达如上所述的IL-15类似物。
进一步地，将编码IL-15类似物的核苷酸序列与原核表达载体相连，转入原核表达宿主菌中，通过诱导表达IL-15类似物。
可选地，IL-15类似物以包涵体方式表达，对包涵体进行复性。
可选地，包涵体溶解于8M尿素溶液中，通过离子交换和反相色谱法纯化。
可选地，原核表达载体为pET41a，表达宿主菌为大肠杆菌BL21(DE3)或C41(DE3)。
本发明的第四个方面提供了一种重组表达载体，其包括编码如上所述的IL-15类似物的核苷酸序列。
本发明的第五个方面提供了一种宿主菌，其转化有编码如上所述的IL-15类似物的核苷酸序列。
本发明的第六个方面提供了一种IL-15类似物的偶联物，IL-15类似物的氨基酸序列包括IL-15氨基酸序列以及在IL-15氨基酸序列的C末端添加的一个或多个氨基酸；所述IL-15类似物的偶联物为IL-15类似物与脂肪酸链连接。
进一步地，所述IL-15类似物的氨基酸序列具有如下特征之一：
1)IL-15-Xa-Yb-Zc，其中，X、Y和Z代表C末端添加的氨基酸序列，a、b和c代表氨基酸个数；X和Z为任意氨基酸或其组合，其中，a和c为0～20；Y为带正电的氨基酸或其组合、或带正电的氨基酸和其他任意氨基酸的组合，其中b为1～7；
2)IL-15-Linker1-Xa-Yb-Zc，其中，Linker代表在IL-15氨基酸序列和C末端添加的氨基酸序列之间的连接序列。
进一步地，Xa包括LPBTG，其中B为任意氨基酸；Linker为(GS)n，n为0～10。
进一步地，脂肪酸链为-(CH2)m-COOH，其中，m为12～19。
进一步地，IL-15类似物与所述脂肪酸链通过体外偶联的方式连接。
进一步地，体外偶联的方式包括：
1)利用酶促反应进行偶联，酶促反应方式的脂肪酸链N末端都带有GGG(氨基酸glycine)；
2)与IL-15类似物中引入的自由半胱氨酸残基进行偶联；
3)与IL-15类似物的N端氨基进行偶联。
进一步地，利用酶促反应进行偶联时，脂肪酸链N带有3个甘氨酸；
与IL-15类似物中引入的自由半胱氨酸残基进行偶联时，脂肪酸链带有马来酰亚胺酯或卤代反应基团；
与IL-15类似物的N端氨基进行偶联时，脂肪酸链带有醛基或琥珀酰亚胺酯官能团。
可选地，在IL-15氨基酸序列的末端添加包括-GS-LPETG的序列。
可选地，脂肪酸链选自：GGG-PEG2-Lys-(CH2)16-COOH和NHS-PEG2-PEG2-γ-Glu-(CH2)17-COOH，GGG-PEG4-PEG4-PEG4-Lys-(CH2)17-COOH、GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH2)17-COOH、HOOC-(CH2)16-γ-Glu-γ-Glu-Lys-GGG、HOOC-(CH2)16-CONH-γ-Glu-γ-Glu-PEG2-Lys-Br、GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)17-COOH、GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)19-COOH、GGG-OEG-C2DA-2OEG-γ-Glu-(CH2)19-COOH、GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH2)19-COOH、CHO-PEG2-PEG2-γ-Glu-(CH2)17-COOH和Mal-C2DA-2OEG-γ-Glu-Tn-(CH2)19-COOH。
本研究发现，在IL-15的C末端添加一些氨基酸有助于IL-15在大肠杆菌中的表达，并且发现带正电荷氨基酸可以明显增强IL-15的表达。本发明的IL-15类似物在大肠杆菌中高表达，表达量约为C端不带额外氨基酸IL-15的20倍，甚至更高。本发明的IL-15类似物主体全部或大部分保留了天然野生型IL-15的氨基酸序列，其体外细胞活性无明显差异，这为IL-15蛋白药物产业化奠定了基础。
本发明是通过体外偶联的方式将脂肪酸链与IL-15类似物相连接，形成IL-15类似物-脂肪酸链偶联产物。由于脂肪酸链是白蛋白的配体，可以与血液中的白蛋白相结合，所以进入体内的IL-15类似物-脂肪酸链形成IL-15类似物-脂肪酸链-白蛋白复合物。一是能使分子量增大，使其逃过肾脏滤过作用；二是通过白蛋白与FcRn结合，并借助其介导的再循环途径避免被胞内溶酶体水解介导的保护机制，从而达到IL-15类似物-脂肪酸链的长效机制。本发明的IL-15类似物的偶联物，没有采用国内外大部分公司所采用的与IL-15受体α亚基共表达形成复合物的药物形式，而是保留了与天然IL-15一样的细胞生物学模式，即完全保留了与IL15Rα受体的结合，来参与信号传导继而发挥功能。
附图说明
图1是本发明一个对比例中野生型IL-15在大肠杆菌中进行表达的SDS-PAGE电泳图。
图2是本发明一个实施例中IL-15类似物在大肠杆菌中进行表达的SDS-PAGE电泳图。其中，图2a是IL-15类似物1～3以及5～8的SDS-PAGE电泳图；图2b是IL-15类似物9～13的SDS-PAGE电泳图；图2c是IL-15类似物4以及类似物14～18的SDS-PAGE电泳图；图2d是IL-15类似物19～27的SDS-PAGE电泳图；图2e是IL-15类似物28～33的SDS-PAGE电泳图；图2f和2g是类似物34～43的SDS-PAGE电泳图，其中类似物38-～43-代表C末端不添加特定的氨基酸。
图3是IL-15以及IL-15类似物1～33的表达量对比图(单位：mg/ml/10OD)
图4是复性并纯化后的IL-15以及IL-15类似物11、18、21、28的SDS-PAGE电泳图。其中，Marker左侧是还原性SDS-PAGE结果，电泳上样缓冲液中添加还原剂2-巯基乙醇；Marker右侧是非还原性SDS-PAGE结果，电泳上样缓冲液中不添加还原剂2-巯基乙醇。
图5是实施例3中RP-UPLC检测IL-15类似物的偶联物纯度色谱图(纯度>95％)。
图6是实施例3中LC-MS鉴定IL-15类似物的偶联物分子量。
图7是实施例4中LC-MS鉴定IL-15类似物的偶联物分子量。
图8是实施例7中IL-15类似物21和IL-15类似物的偶联物在小鼠体内的抑瘤效果图。
具体实施方式
以下将结合实施例对本发明作进一步地说明，应理解这些实施例仅作为例证的目的，不用于限制本发明的保护范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。所采用的试剂，若无特殊说明，均为市售或公开渠道可以获得的试剂。
本发明的具体实施方式中，带正电的氨基酸为亮氨酸(Lys，K)、精氨酸(Arg，R)、组氨酸(His，H)。
作为改造基础的IL-15的氨基酸序列选自：
1)如SEQ ID NO.1所示氨基酸序列；
2)与SEQ ID NO.1所示的氨基酸序列通过一个或多个氨基酸取代、缺失或添加衍生产生的氨基酸序列；
3)与SEQ ID NO.1具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列
本发明的具体实施方式中，在IL-15的C端增加一个或多个氨基酸，尤其是带正电的氨基酸，能够提高IL-15的表达，尤其是在原核表达系统中的表达。
在一些具体实施方式中，IL-15类似物的氨基酸序列可以通过以下通式来表达：IL-15-Xa-Yb-Zc，即在IL-15的C端添加了氨基酸序列Xa-Yb-Zc。其中，X、Y和Z代表C末端添加的氨基酸序列，a、b和c代表氨基酸个数。X和Z为任意氨基酸或其组合，其中，a和c为0～20；Y为带正电的氨基酸或其组合、或带正电的氨基酸和其他任意氨基酸的组合，其中b为1～7。
在一些具体实施方式中，IL-15类似物的氨基酸序列可以通过以下通式来表达：IL-15-Linker-Xa-Yb-Zc，即在IL-15的C端添加了一个Linker序列，以及氨基酸序列Xa-Yb-Zc。其中，X、Y和Z代表C末端添加的氨基酸序列，a、b和c代表氨基酸个数。X和Z为任意氨基酸或其组合，其中，a和c为0～20；Y为带正电的氨基酸或其组合、或带正电的氨基酸和其他任意氨基酸的组合，其中b为1～7。
在一些可选的具体实施方式中，Linker可以为(GGGGS)n、(GS)n或(GAPQ)n，n为1～5。
在一些具体实施方式中，IL-15类似物的氨基酸序列的上述通式中Xa包括LPBTG，其中，B为任意氨基酸，Linker为(GS)n，n为0～10。在一个具体实施方式中，该IL-15类似物为在IL-15氨基酸序列的末端添加包括-GS-LPETG的序列。
在一些具体实施方式中，为了提高IL-15类似物的长效性，将IL-15类似物在体外与脂肪酸链进行偶联。脂肪酸链体外偶联的方式可以是：
1)和N端氨基的偶联，例如用醛基或琥珀酰亚胺酯等。
2)定点与引入的自由半胱氨酸残基偶联，例如在IL-15类似物C末端引入一个自由的半胱氨酸残基，利用马来酰亚胺基团或是卤代基团。
3)利用酶促反应，例如Sortase A酶可以利用IL-15C末端带有的小肽LPETG，将N端带有GGG的脂肪酸链与之偶联(Bentley ML等(2008).J Biol Chem 283:14762-14771)。脂肪酸链结构可以包括表1所示的结构。
表1脂肪酸链结构
*上表中，LPET表示IL-15的C端添加的氨基酸序列包括LPET。
实施例1野生型IL-15及IL-15类似物在大肠杆菌中的表达
1.1表达载体的构建
野生型IL-15核苷酸序列由上海生工公司合成。
(1)引物设计
通过在反向引物中引入需要添加的不同氨基酸的碱基序列来改变IL-15的C端序列。野生型IL-15、构建的IL-15类似物的氨基酸序、核苷酸序列和构建时使用的引物序列如表2所示：
表2野生型IL-15、IL-15类似物的氨基酸序、核苷酸序列和构建时使用的引物序列
类似物34～43是在野生型IL-15上引入突变形成IL-15突变体后，在IL-15突变体的基础上在C末端添加序列GSLPETGGSGGSHHHHHH形成的，类似物34-～43-就是引入突变后形成的IL-15突变体。突变类似物34～43以及C端不添加GSLPETGGSGGSHHHHHH序列的类似物38-～类似物43-的核苷酸序列由金唯智公司合成并克隆至pET41a载体。
(2)PCR扩增
分为三种情况
第一种为只需一个反向引物，共1轮PCR，加样体系：50μL(配置1管)引物：正向引物和反向引物；模板：IL-15；退火温度：61℃；延伸时间：30s
第二种为需两个反向引物，共2轮PCR
第1轮PCR，加样体系：20μL(配置1管)
引物：正向引物和反向引物1；模板：IL-15；退火温度：61℃；延伸时间：30s
第2轮PCR，加样体系：50μL(配置1管)
引物：正向引物和反向引物2；模板：第一轮PCR产物；退火温度：61℃；延伸时间：30s
第三种为三个反向引物，共3轮PCR
第1轮PCR，加样体系：20μL(配置1管)
引物：正向引物和反向引物1；模板：IL-15；退火温度：61℃；延伸时间：30s
第2轮PCR，加样体系：20μL(配置1管)
引物：正向引物和反向引物2；模板：第1轮PCR产物；退火温度：61℃；延伸时间：30s
第3轮PCR，加样体系：50μL(配置1管)
引物：正向引物和反向引物3；模板：第2轮PCR产物；退火温度：61℃；延伸时间：30s
PCR加样体系：20μL
PCR加样体系：50μL
PCR反应程序：
(3)载体酶切
反应体系：30μL，混匀后37℃过夜
(4)胶回收PCR产物和酶切后载体
(5)重组进载
反应体系：20μL
载体与片段摩尔比为2:1，37℃反应30min，立即置于冰上冷却，转化入DH5α中。
(6)菌检
在1.5mL的离心管中加入0.5mL含Amp抗性的LB培养基，向其中接种长得良好的单菌落，一共接种5管，在37℃摇床中进行振荡培养。培养3h以后，取1μL做模板，进行菌液PCR。
PCR反应体系：10μL
PCR反应程序：
PCR完成后用琼脂糖凝胶电泳进行检测，选择3个阳性克隆进行测序(生工)。
(7)阳性菌液保存及质粒的提取
将测序正确的阳性克隆接种于5mL Amp抗性的LB液体培养基中，接种量为10μL。取500μL的菌液加入1.5mL的离心管中，加入500μL 40％的甘油，用于保存。写好名称，宿主菌，日期，封上封口膜放入-80℃冰箱保存。
剩余的菌液离心收集菌体，用于质粒提取。
1.2野生型IL-15和IL-15类似物的蛋白表达
(1)转化BL21(DE3)
a)取2uL质粒加入100uL BL21(DE3)感受态细胞中，立即混匀，冰上放置30min；
b)42℃热激90s，迅速冰浴2min；
c)加入500uL LB培养基，37℃，rpm<＝200振荡培养60min；
d)6000rpm离心1min，弃去大部分上清，留约100-150uL，重悬菌体后，涂于含Amp的LB平板上，37℃过夜培养。
(2)小量表达
a)保菌：挑取1个单克隆于1mL Amp抗性的LB培养基中，37℃，220rpm振荡培养约5h，加入1mL 40％甘油，分装2管，冻存于-80℃；
b)在上一步管中加入2.5mL含Amp的LB液体培养基，37℃，220rpm振荡培养过夜；
c)将过夜培养的菌液按1:50比例转接到20ml含Amp的LB培养基中，37℃，220rpm，培养至OD600＝0.6(约3h)，加入终浓度0.5mM IPTG，37℃，220rpm培养3h；
(3)SDS-PAGE鉴定表达量
a)测定培养液的OD600，取10OD菌液，10000rpm，2min离心，去掉上清；
b)用1mL裂解液(10mM Tris-HCl,pH 8.0)重悬菌体，置于冰上进行超声裂解细胞，超声条件：130W、4min、on 3s、off 3s；
c)超声结束后，取80uL标记为T(lysis total)，剩余液体12000rpm,10min离心得到上清(lysis supernatant，S)和沉淀(lysis pellet，P)；80uL T、P和S分别加入20uL 5×Reduce loading buffer，95℃加热5min，每个样品取12.5uL(0.1OD)进行SDS-PAGE电泳。
IL-15野生型的电泳图如图1所示，无论是总裂解液、上清或沉淀中，都基本检测不到野生型IL-15的表达；IL-15类似物1～类似物33的电泳图如图2a～图2e所示，IL-15类似物表达量要明显高于野生型IL-15；而C端带有GSLPETGGSGGSHHHHHH序列的IL-15类似物(类似物34～43)表达量也要明显高于C端不带序列的IL-15类似物(类似物38-～43-)，如图2f和2g所示。
(4)HPLC鉴定表达量
a)表达后收取10OD细胞，用1ml，10mM Tris-HCl，pH8.0的buffer重悬；
b)超声破碎，条件同跑胶样品处理(上述(3)中)的条件；
c)破碎后样品，12000rpm，离心10min，弃上清；
d)沉淀用1ml新鲜配制的8M Urea/10mM Tris-HCl，pH8.0,10mM DTT室温振荡溶解约1h；
e)用0.2um滤头过滤，上样进行高效液相色谱分析。利用C4分析柱，以去离子水含0.1％TFA为流动相A，以乙腈含0.1％TFA为流动相B，然后用15分钟从20％B到60％B的梯度来进行分析；
HPLC定量结果如图3所示，IL-15类似物1～33表达量要高于野生型IL-15的表达量20倍左右。
实施例2 IL-15类似物体外细胞活性实验
2.1IL-15类似物的准备
包涵体表达的IL-15类似物11、类似物18、类似物21及类似物28溶解在8M尿素溶液中，通过离子交换和反相色谱法纯化(具体方法参见：Yunieret al,Preparative Biochemistry and Biotechnology,47:9,889-900)，纯化得到较纯的蛋白，SDS-PAGE电泳图如图4所示。
2.2CTLL-2细胞实验
CTLL-2细胞的增殖实验是普遍应用的细胞水平测定白介素刺激免疫细胞活性的实验。因此，此处通过野生型IL-15和IL-15类似物对CTLL-2细胞的增殖效果来查看IL-15类似物的生物活性。
1)CTLL-2细胞准备：将细胞用含有FBS和Rat-T-Stim的培养液重悬起来。
2)加样：将细胞接种于96孔培养板，每孔0.1ml。同时将待测样品IL-15类似物11、18、21和28的蛋白(即2.1中制备的蛋白)分别做倍比稀释，每孔加入0.1ml，每个稀释浓度均设3复孔。另设培养液对照孔(100ul细胞+100ul培养液)。37度，5％CO2孵育72小时。
3)MTS加入：每孔加入20μlAQueous One Solution Reagent，37度，5％CO2孵育2～4小时。
4)测定：用酶标测定仪于波长490nm测吸光值(A)并计算EC50值。
结果如表3所示，IL-15类似物和野生型IL-15的细胞活性无明显差别。
表3CTLL-2细胞活性实验
| EC50(ng/ml) | |
| IL-15 | 0.05445 |
| 类似物11 | 0.05252 |
| 类似物18 | 0.05609 |
| 类似物21 | 0.05235 |
| 类似物28 | 0.05085 |
实施例3IL-15类似物的偶联物的制备(酶促反应法)
利用纯化后的IL-15类似物21(IL-15-GS-LPETG-GSGGSHHHHHH)，通过转肽酶SortaseA酶催化的连接反应将N端带有GGG的脂肪酸链(816366)与IL-15-GS-LPETG-GSGGSHHHHHH相连接。反应按照Sortase A:IL-15:脂肪酸链比例为1:6:30进行，反应buffer为50mM Tris-HCl,1mM CaCl2，150mM NaCl pH8.0在室温下反应3小时后，进行纯化。纯化采用反相色谱C8(赛分科技)将未偶联的IL-15类似物21，未反应的脂肪酸链以及偶联后的产物分开，最终产物经过UPLC检测纯度(如图5)和LC-MS(图6)鉴定表明，IL-15类似物21已偶联上脂肪酸链，形成IL-15类似物21-816366。
实施例4 IL-15类似物的偶联物的制备(IL-15类似物N端氨基偶联脂肪酸链)
利用纯化后的IL-15类似物21通过带有的琥珀酰亚胺酯的脂肪酸链(820044)在中性pH下与N端的氨基进行偶联。反应按照IL-15:脂肪酸链比例为1:1进行，反应buffer为PBS，pH7.2在室温下反应1小时后，进行纯化。纯化采用反相色谱C8(赛分科技)将未偶联的IL-15，未反应的脂肪酸链以及偶联后的产物分开，最终产物经过LC-MS鉴定表明，如图7所示，IL-15已偶联上脂肪酸链，形成IL-15类似物21-820044。
实施例5 IL-15类似物21与IL-15类似物的偶联物和IL15Rα受体的结合实验
本实施例中使用的IL-15类似物的偶联物为实施例3中制备的。
本实施例中采用生物膜干涉技术(biolayer interferomeory，BLI)进行测定目的蛋白与受体间的亲和力，方法可参见(Estep,P等人，High throughput solution Basedmeasurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。实验所用受体蛋白IL15Rα-his为北京志道生物科技有限公司生产。缓冲液配方为10mM HEPES，150mM NaCl，3mM EDTA和0.05％tween20；受体IL15Rα-his是预先固定化在HISIK传感器(Pall Fortebio公司，货号#18-5120)上的，然后按照建立的方法，以baseline，loading，baseline，Association和Dissociation的步骤进行。数据获取和分析工作分别采用Octet RED96软件Data acquisition 11.0和Data analysis 11.0进行。
IL-15类似物与IL-15类似物的脂肪酸链偶联产物与IL15Rα受体的亲和力如表4所示，相比偶联前的IL-15类似物，IL-15类似物的脂肪酸链偶联产物与IL15Rα受体的亲和力没有明显变化。
表4亲和力实验结果表
| 蛋白名称 | 与IL15Rα的亲和力(M) |
| IL-15类似物21 | 1.18E-09 |
| IL-15类似物21-816366 | 6.6E-09 |
| IL-15类似物21-823479 | 4E-09 |
| IL-15类似物21-823480 | 8E-10 |
实施例6 IL-15类似物21与IL-15类似物的偶联物在小鼠体内的半衰期本实施例中使用的IL-15类似物的偶联物为实施例3中制备的。
8只C57BL/6小鼠，分为2组，每组4只，每个时间点采2只，循环采血。小鼠尾静脉注射IL-15，0.5mg/kg，给药后立即取样、10min、30min、1.5h、4h取血；小鼠尾静脉注射长效IL-15，0.5mg/kg，给药后立即取样、1h、2h、4h取血，每个点眼眶取血50-100μl。取血清进行IL-15ELISA检测。
根据半衰期计算公式：t1/2＝0.693/k，k＝(lnc0-lnc)/t可以得到IL-15的半衰期大约为5min，而长效IL-15可以达到1.5h。
实施例7 IL-15类似物21和IL-15类似物的偶联物在小鼠体内肿瘤抑制实验
本实施例中使用的IL-15类似物的偶联物为实施例3中制备的。
15只6～8周龄的C57BL/6小鼠，随机分为3组，分别为试剂对照组(PBS)、IL-15组和长效IL-15组，每组5只小鼠。Day1，小鼠靠颈背部皮下接种B16-F10细胞，2×105/100μL/只。在Day4-Day8，PBS组和IL-15组分别以静脉注射(i.v.)的方式连续给药5天，给药量为20μg/100μL/只或100μL/只(PBS组)。而IL-15类似物的偶联物组在Day4和Day7以同样给药方式给药两次，每次相同给药量为20μg/100μL/只。在Day10开始观察肿瘤大小，持续观察6天。
结果如图8所示，和对照组相比，IL-15和长效IL-15均具有明显的抑瘤作用。
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Leu Ile Gln Ser Val His Ile Arg Gly Asp Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Gly Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Gln Gln Ser Leu Ser Ser Asn Ala
65 70 75 80
Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
100 105 110
Ile Asn Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 7
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 7
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp
1 5 10 15
Leu Ile Gln Ser Val His Ile Arg Gly Asp Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Gly Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Gln Gln Ser Leu Ser Ser Asn Ala
65 70 75 80
Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Ser Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Val Phe
100 105 110
Ile Asn Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 8
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 8
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Arg Lys Ile Arg Gly
1 5 10 15
Asp Ile Gln Ser Val His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Arg Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Asn Gln Ser Leu Ser Ser Asn Ala
65 70 75 80
Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Leu Phe
100 105 110
Ile Gln Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 9
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 9
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Arg Lys Ile Arg Gly
1 5 10 15
Asp Leu Asn Ala Val His Val Asp Ala Thr Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Arg Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Asn Gln Ser Leu Ser Ser Asn Ala
65 70 75 80
Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Leu Phe
100 105 110
Ile Gln Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 10
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 10
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Arg Lys Ile Arg Gly
1 5 10 15
Asp Ile Gln Ser Val His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Arg Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Asn Gln Ser Leu Ala Ala Gln Ala
65 70 75 80
Gln Leu Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Leu Phe
100 105 110
Ile Gln Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 11
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 11
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Arg Lys Ile Arg Gly
1 5 10 15
Asp Leu Asn Ala Val His Val Asp Ala Thr Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Arg Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr
50 55 60
Val Glu Asn Leu Ile Ile Leu Ala Asn Gln Ser Leu Ala Ala Gln Ala
65 70 75 80
Gln Leu Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Leu Phe
100 105 110
Ile Gln Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 12
<211> 134
<212> PRT
<213> Artificial Sequence (人工序列)
<400> 12
Met Ser Asn Trp Val Asn Val Ile Ser Asp Leu Arg Lys Ile Arg Gly
1 5 10 15
Asp Ile Gln Ser Val His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp
20 25 30
Val His Pro Ser Cys Arg Val Thr Ala Met Lys Cys Phe Leu Leu Glu
35 40 45
Leu Gln Leu Ile Ser Leu Asp Ser Gly Asp Ala Ser Ile His Glu Thr
50 55 60
Val Glu Gln Leu Ile Leu Leu Ala Asn Gln Ser Leu Ala Ala Gln Ala
65 70 75 80
Gln Leu Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys
85 90 95
Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Leu Phe
100 105 110
Ile Gln Thr Ser Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser
115 120 125
His His His His His His
130
<210> 13
<211> 396
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 13
atgtgggtga acgttatcag cgacctgaag aaaatcgagg atctgattca gagcatgcac 60
attcgtggtg acctgtacac cgaaagcgat gtgcacccga gctgcaaggt taccgcgatg 120
aaatgcttcc tgctggagct gcaagtgatc agcctggaaa gcggtgacgc gagcattcac 180
gataccgttg agaacctgat cattctggcg aacaacagcc tgagcagcaa cgcgaacgtg 240
accgagagcg gctgcaagga atgcgaggaa ctggaggaaa agaacatcaa agaattcctg 300
cagagctttg tgcacatcgt tcaaatgttt attaacacca gcggttctct gccggaaacc 360
ggtggttctg gtggttctca ccaccaccac caccac 396
<210> 14
<211> 402
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 14
atgagcaact gggtgaacgt tatcagcgac ctgaagaaaa tcgaggatct gattcagagc 60
atgcacattg acgcgaccct gtacaccgaa agcgatgtgc acccgagctg caaggttacc 120
gcgatgaaat gcttcctgct ggagctgcaa gtgatcagcc tggaaagcgg tgacgcgagc 180
attcacgata ccgttgagaa cctgatcatt ctggcgaaca acagcctgag cagcaacgcg 240
aacgtgaccg agagcggctg caaggaatgc gaggaactgg aggaaaagaa catcaaagaa 300
ttcctgcaga gctttgtgca catcgttcaa atgtttatta acaccagcgg ttctctgccg 360
gaaaccggtg gttctggtgg ttctcaccac caccaccacc ac 402
<210> 15
<211> 402
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 15
atggccaact gggtgaacgt tatcagcgac ctgaagaaaa tcgaggatct gattcagagc 60
atgcacattg acgcgaccct gtacaccgaa agcgatgtgc acccgagctg caaggttacc 120
gcgatgaaat gcttcctgct ggagctgcaa gtgatcagcc tggaaagcgg tgacgcgagc 180
attcacgata ccgttgagaa cctgatcatt ctggcgaaca acagcctgag cagcaacgcg 240
aacgtgaccg agagcggctg caaggaatgc gaggaactgg aggaaaagaa catcaaagaa 300
ttcctgcaga gctttgtgca catcgttcaa atgtttatta acaccagcgg ttctctgccg 360
gaaaccggtg gttctggtgg ttctcaccac caccaccacc ac 402
<210> 16
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 16
atgagcaact gggtgaatgt gattagtgat ctgaaaaaaa ttgaagatct gattcagagt 60
gtgcatatta gaggtgatct gtatactgaa agtgatgtgc atccaagctg caaagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatggcagc 180
attcatgata ctgtggaaaa cctgattatt ctggcacagc agagcctgag cagcaatgcc 240
aatgtgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcaa atgtttatta acactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 17
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 17
atgagcaact gggtgaatgt gattagtgat ctgaaaaaaa ttgaagatct gattcagagt 60
gtgcatatta gaggtgatct gtatactgaa agtgatgtgc atccaagctg caaagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatggcagc 180
attcatgata ctgtggaaaa cctgattatt ctggcacagc agagcctgag cagcaatgcc 240
aatgtgactg aaagtggctg caaagaatgt gaagaactga gtgaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcaa gtgtttatta acactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 18
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 18
atgagcaact gggtgaatgt gattagtgat ctgagaaaaa ttagaggtga tattcagagt 60
gtgcatattg atgccaccct gtatactgaa agtgatgtgc atccaagctg cagagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatgcaagc 180
attcatgata ctgtggaaaa cctgattatt ctggccaatc agagcctgag cagcaatgcc 240
aatgtgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcag ctgtttattc agactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 19
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 19
atgagcaact gggtgaatgt gattagtgat ctgagaaaaa ttagaggtga tctgaatgca 60
gtgcatgtgg atgccaccct gtatactgaa agtgatgtgc atccaagctg cagagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatgcaagc 180
attcatgata ctgtggaaaa cctgattatt ctggccaatc agagcctgag cagcaatgcc 240
aatgtgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcag ctgtttattc agactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 20
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 20
atgagcaact gggtgaatgt gattagtgat ctgagaaaaa ttagaggtga tattcagagt 60
gtgcatattg atgccaccct gtatactgaa agtgatgtgc atccaagctg cagagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatgcaagc 180
attcatgata ctgtggaaaa cctgattatt ctggccaatc agagcctggc agcccaagca 240
cagctgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcag ctgtttattc agactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 21
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 21
atgagcaact gggtgaatgt gattagtgat ctgagaaaaa ttagaggtga tctgaatgca 60
gtgcatgtgg atgccaccct gtatactgaa agtgatgtgc atccaagctg cagagtgact 120
gccatgaaat gctttctgct ggaactgcaa gtgattagcc tggaaagtgg tgatgcaagc 180
attcatgata ctgtggaaaa cctgattatt ctggccaatc agagcctggc agcccaagca 240
cagctgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcag ctgtttattc agactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 22
<211> 402
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 22
atgagcaact gggtgaatgt gattagtgat ctgagaaaaa ttagaggtga tattcagagt 60
gtgcatattg atgccaccct gtatactgaa agtgatgtgc atccaagctg cagagtgact 120
gccatgaaat gctttctgct ggaactgcag ctgattagcc tggatagtgg tgatgcaagc 180
attcatgaaa ctgtggaaca gctgattctg ctggccaatc agagcctggc agcccaagca 240
cagctgactg aaagtggctg caaagaatgt gaagaactgg aagaaaaaaa cattaaagaa 300
tttctgcaga gctttgtgca tattgtgcag ctgtttattc agactagtgg cagcctgcct 360
gaaactggtg gcagtggtgg cagccatcat catcatcatc at 402
<210> 23
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 23
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 24
<211> 58
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 24
gtggtggtgg tggtgagaac cagaaccaga accgctggtg ttaataaaca tttgaacg 58
<210> 25
<211> 48
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 25
gtggtggtgg tggtgctcga gttagtggtg gtggtggtgg tgagaacc 48
<210> 26
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 26
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 27
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 27
gtggtggtgg tggtggtgag aaccgctggt gttaataaac atttgaacg 49
<210> 28
<211> 44
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 28
gtggtggtgg tggtgctcga gttagtggtg gtggtggtgg tgag 44
<210> 29
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 29
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 30
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 30
acgctttttg gtgctagcaa gtgggctggt gttaataaac atttgaacg 49
<210> 31
<211> 45
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 31
gtggtggtgg tggtgctcga gttaacgctt tttggtgcta gcaag 45
<210> 32
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 32
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 33
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 33
cttcttttta gcagactttg gaaggctggt gttaataaac atttgaacg 49
<210> 34
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 34
gtggtggtgg tggtgctcga gttacttctt tttagcagac tttggaagg 49
<210> 35
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 35
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 36
<211> 46
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 36
cttctttttt ttcttctttt tgctggtgtt aataaacatt tgaacg 46
<210> 37
<211> 50
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 37
gtggtggtgg tggtgctcga gttacttctt ttttttcttc tttttgctgg 50
<210> 38
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 38
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 39
<211> 59
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 39
attccacaag ctgtggagca ccctgtggag caccgctggt gttaataaac atttgaacg 59
<210> 40
<211> 59
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 40
gtggtggtgg tggtgctcga gttaatgatg gtgtgcagat tccacaagct gtggagcac 59
<210> 41
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 41
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 42
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 42
tttgtggtga gcgctagata caaggctacc gctggtgtta ataaacattt gaacg 55
<210> 43
<211> 47
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 43
gtggtggtgg tggtgctcga gttatttgtg gtgagcgcta gatacaa 47
<210> 44
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 44
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 45
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 45
tttgcgacgg tggtgttcga taaggctacc gctggtgtta ataaacattt gaacg 55
<210> 46
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 46
gtggtggtgg tggtgctcga gttatttgcg acggtggtgt tcg 43
<210> 47
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 47
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 48
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 48
ctttttctta cggtgttcta caatgctacc gctggtgtta ataaacattt gaacg 55
<210> 49
<211> 50
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 49
gtggtggtgg tggtgctcga gttacttttt cttacggtgt tctacaatgc 50
<210> 50
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 50
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 51
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 51
acggcgacga ccagtttttg gtacgctacc gctggtgtta ataaacattt gaacg 55
<210> 52
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 52
gtggtggtgg tggtgctcga gttaacggcg acgaccagtt ttt 43
<210> 53
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 53
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 54
<211> 52
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 54
ctttttaccg ctagcaacca ggctaccgct ggtgttaata aacatttgaa cg 52
<210> 55
<211> 46
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 55
gtggtggtgg tggtgctcga gttacttttt accgctagca accagg 46
<210> 56
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 56
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 57
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 57
atgatggtga ccagatttac gatggctacc gctggtgtta ataaacattt gaacg 55
<210> 58
<211> 48
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 58
gtggtggtgg tggtgctcga gttaatgatg gtgaccagat ttacgatg 48
<210> 59
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 59
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 60
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 60
cttgtgacga ccagtttttg gaaggctacc gctggtgtta ataaacattt gaacg 55
<210> 61
<211> 46
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 61
gtggtggtgg tggtgctcga gttacttgtg acgaccagtt tttgga 46
<210> 62
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 62
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 63
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 63
atggcgacga ccagtcttct ttttgctggt gttaataaac atttgaacg 49
<210> 64
<211> 45
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 64
gtggtggtgg tggtgctcga gttaatggcg acgaccagtc ttctt 45
<210> 65
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 65
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 66
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 66
cttatgacga ccagaacgtg gaaggctggt gttaataaac atttgaacg 49
<210> 67
<211> 46
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 67
gtggtggtgg tggtgctcga gttacttatg acgaccagaa cgtgga 46
<210> 68
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 68
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 69
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 69
gtgatgatgg tgagtttcaa ccaggctggt gttaataaac atttgaacg 49
<210> 70
<211> 48
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 70
gtggtggtgg tggtgctcga gttagtgatg atggtgagtt tcaaccag 48
<210> 71
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 71
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 72
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 72
atgatggtga gtttctggac gaacgctggt gttaataaac atttgaacg 49
<210> 73
<211> 47
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 73
gtggtggtgg tggtgctcga gttaatgatg gtgagtttct ggacgaa 47
<210> 74
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 74
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 75
<211> 34
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 75
cttttttttg ctggtgttaa taaacatttg aacg 34
<210> 76
<211> 50
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 76
gtggtggtgg tggtgctcga gttacttttt tttgctggtg ttaataaaca 50
<210> 77
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 77
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 78
<211> 40
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 78
atgatgatgg tgacggctgg tgttaataaa catttgaacg 40
<210> 79
<211> 44
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 79
gtggtggtgg tggtgctcga gttaatgatg atggtgacgg ctgg 44
<210> 80
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 80
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 81
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 81
atgatgatgg tgagtttcac gcttgctggt gttaataaac atttgaacg 49
<210> 82
<211> 47
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 82
gtggtggtgg tggtgctcga gttaatgatg atggtgagtt tcacgct 47
<210> 83
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 83
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 84
<211> 52
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 84
accaccagaa ccaccggttt ccggcagaga accgctggtg ttaataaaca tt 52
<210> 85
<211> 57
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 85
gtggtggtgg tggtgctcga gttagtggtg gtggtggtgg tgagaaccac cagaacc 57
<210> 86
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 86
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 87
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 87
cttcttttta ccagtttcaa ggtggctggt gttaataaac atttgaacg 49
<210> 88
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 88
gtggtggtgg tggtgctcga gttacttctt tttaccagtt tcaaggtgg 49
<210> 89
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 89
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 90
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 90
acggcgacga ccagattcaa catggctggt gttaataaac atttgaacg 49
<210> 91
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 91
gtggtggtgg tggtgctcga gttaacggcg acgaccagat tca 43
<210> 92
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 92
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 93
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 93
cttcttttta ccagtatgac gacggctggt gttaataaac atttgaacg 49
<210> 94
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 94
gtggtggtgg tggtgctcga gttacttctt tttaccagta tgacgacgg 49
<210> 95
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 95
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 96
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 96
atgatggtga ccagtcttaa catggctggt gttaataaac atttgaacg 49
<210> 97
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 97
gtggtggtgg tggtgctcga gttaatgatg gtgaccagtc ttaacatgg 49
<210> 98
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 98
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 99
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 99
atgatgacga ccagacttaa catggctggt gttaataaac atttgaacg 49
<210> 100
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 100
gtggtggtgg tggtgctcga gttaatgatg acgaccagac ttaacatgg 49
<210> 101
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 101
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 102
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 102
gtgacgatgg ctagacttaa catggctggt gttaataaac atttgaacg 49
<210> 103
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 103
gtggtggtgg tggtgctcga gttagtgacg atggctagac ttaacatgg 49
<210> 104
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 104
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 105
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 105
gctaccgcta ccgctaccgc taccgctacc gctggtgtta ataaacattt gaacg 55
<210> 106
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 106
atgatggtga ccagacttta ccaggctacc gctaccgcta ccg 43
<210> 107
<211> 48
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 107
gtggtggtgg tggtgctcga gttaatgatg gtgaccagac tttaccag 48
<210> 108
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 108
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 109
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 109
cttatggtga ccgctctttg gacggctggt gttaataaac atttgaacg 49
<210> 110
<211> 46
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 110
gtggtggtgg tggtgctcga gttacttatg gtgaccgctc tttgga 46
<210> 111
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 111
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 112
<211> 58
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 112
gtggtggtgg tggtgctcga gttaacactt tttgctggtg ttaataaaca tttgaacg 58
<210> 113
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 113
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 114
<211> 49
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 114
atggtgttta ccagccttgt gaaggctggt gttaataaac atttgaacg 49
<210> 115
<211> 47
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 115
gtggtggtgg tggtgctcga gttaatggtg tttaccagcc ttgtgaa 47
<210> 116
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 116
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 117
<211> 52
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 117
gtggtggtgg tggtgctcga gttatttgct ggtgttaata aacatttgaa cg 52
<210> 118
<211> 43
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 118
taagaaggag atatacatat gaactgggtg aacgttatca gcg 43
<210> 119
<211> 55
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 119
gtggtggtgg tggtgctcga gttacttttt gctggtgtta ataaacattt gaacg 55
Claims (13)
1.一种IL-15类似物，其特征在于，所述IL-15类似物的氨基酸序列由IL-15氨基酸序列以及在IL-15氨基酸序列的C末端添加的一个或多个氨基酸组成，所述一个或多个氨基酸包括带正电的氨基酸；
所述IL-15类似物的氨基酸序列具有如下特征：IL-15-Xa-Yb-Zc，其中，X、Y和Z代表C末端添加的氨基酸序列，a、b和c代表氨基酸个数；
所述X包括V、I、P、L、E、A、S、C、T、G中的任一个或其组合，Z为任意氨基酸或其组合，其中，a和c为0~20；Y为带正电的氨基酸H、R、K或其组合，其中b为1~7；
在所述IL-15的C末端添加的一个或多个氨基酸如下所示：GSGSGS-HHHHHH、GS-HHHHHH、PLASTKKR、LPKSAKKK、KKKKKKK、(GAPQGAPQ)-LVESAHHH、GS-LVSSAHHK、GS-LIEHHRRK、GS-IVEHRKKK、GS-VPKTGRRR、GS-LVASGKK、GS-HRKSGHHH、GS-LPKTGRHK、KKKTGRRH、LPRSGRHK、LVETHHHH、VRPETHHH、KKK、RHHHH、KRETHHHH、GS-LPETGGSGGSHHHHHH、HLETGKKK、HVESGRRR、RRHTGKKK、HVKTGHHH、HVKSGRHH、HVKSSHRH、GSGSGSGSGS-LVKSGHHH、RPKSGHHK、KKC、LHKAGKHH、K、KK；
IL-15的氨基酸序如SEQ ID NO. 1所示。
2.一种编码如权利要求1所述的IL-15类似物的核苷酸序列。
3.一种IL-15类似物的制备方法，其特征在于，在原核系统中表达如权利要求1所述的IL-15类似物。
4.如权利要求3所述的IL-15类似物的制备方法，其特征在于，将编码IL-15类似物的核苷酸序列与原核表达载体相连，转入原核表达宿主菌中，通过诱导表达所述IL-15类似物。
5.一种重组表达载体，其特征在于，包括编码如权利要求1所述的IL-15类似物的核苷酸序列。
6.一种宿主菌，其特征在于，转化有编码如权利要求1所述的IL-15类似物的核苷酸序列。
7.一种IL-15类似物的偶联物，其特征在于，IL-15类似物的氨基酸序列包括IL-15氨基酸序列以及在IL-15氨基酸序列的C末端添加的一个或多个氨基酸，其中IL-15类似物为如权利要求1所述的IL-15类似物；所述IL-15类似物的偶联物为IL-15类似物与脂肪酸链连接。
8.如权利要求7所述的IL-15类似物的偶联物，其特征在于，所述脂肪酸链为-(CH2)m-COOH，其中，m为12~19。
9.如权利要求7所述的IL-15类似物的偶联物，其特征在于，所述IL-15类似物与所述脂肪酸链通过体外偶联的方式连接。
10.如权利要求9所述的IL-15类似物的偶联物，其特征在于，所述体外偶联的方式包括：
1）利用酶促反应进行偶联，酶促反应方式的脂肪酸链N末端都带有GGG；
2）与IL-15类似物中引入的自由半胱氨酸残基进行偶联；
3）与IL-15类似物的N端氨基进行偶联。
11.如权利要求10所述的IL-15类似物的偶联物，其特征在于，
利用酶促反应进行偶联时，脂肪酸链末端带有3个甘氨酸；
与IL-15类似物中引入的自由半胱氨酸残基进行偶联时，脂肪酸链带有马来酰亚胺酯或卤代反应基团；
与IL-15类似物的N端氨基进行偶联时，脂肪酸链带有醛基或琥珀酰亚胺酯官能团。
12.如权利要求7所述的IL-15类似物的偶联物，其特征在于，所述IL-15类似物为在IL-15氨基酸序列的末端添加包括-GS-LPETG的序列。
13.如权利要求7所述的IL-15类似物的偶联物，其特征在于，所述脂肪酸链选自：GGG-PEG2-Lys-(CH2)16-COOH和NHS-PEG2-PEG2-γ-Glu-(CH2)17-COOH、GGG-PEG4-PEG4-PEG4-Lys-(CH2)17-COOH、GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH2)17-COOH、HOOC-(CH2)16-γ-Glu-γ-Glu-Lys-GGG、HOOC-(CH2)16-CONH- γ -Glu- γ -Glu-PEG2-Lys-Br、GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)17-COOH、GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)19-COOH、GGG-OEG-C2DA-2OEG-γ-Glu-(CH2)19-COOH、GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH2)19-COOH、CHO-PEG2-PEG2-γ-Glu-(CH2)17-COOH和Mal-C2DA-2OEG-γ-Glu-Tn-(CH2)19-COOH。
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| CN2019109101585 | 2019-09-25 | ||
| CN201910910158 | 2019-09-25 |
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| US (1) | US20220348624A1 (zh) |
| EP (1) | EP4015526A4 (zh) |
| JP (1) | JP2022550743A (zh) |
| KR (1) | KR20220069018A (zh) |
| CN (1) | CN112552391B (zh) |
| AU (1) | AU2020354198A1 (zh) |
| CA (1) | CA3153542A1 (zh) |
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| JP2024524891A (ja) * | 2021-06-23 | 2024-07-09 | サイチューン ファーマ | インターロイキン－１５に基づく免疫サイトカイン |
| CN117597355A (zh) * | 2021-06-23 | 2024-02-23 | 赛腾制药 | 白细胞介素15变体 |
| CN113754752B (zh) * | 2021-09-02 | 2023-11-14 | 惠誉生物医学(中山)研究院有限公司 | 一种多肽及其制备方法与应用 |
| CN113956346B (zh) * | 2021-10-26 | 2024-03-01 | 西安龙腾景云生物科技有限公司 | 一种重组白介素-15变体 |
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|---|---|---|---|---|
| CN1921879A (zh) * | 2003-12-29 | 2007-02-28 | 明斯特大学临床医学院 | 由il－15刺激和活化毛发生长的方法 |
| CN108147990A (zh) * | 2016-12-02 | 2018-06-12 | 上海中医药大学 | 一种膜锚定元件及其应用 |
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-
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- 2020-09-24 KR KR1020227010181A patent/KR20220069018A/ko unknown
- 2020-09-24 CA CA3153542A patent/CA3153542A1/en active Pending
- 2020-09-24 US US17/763,227 patent/US20220348624A1/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1921879A (zh) * | 2003-12-29 | 2007-02-28 | 明斯特大学临床医学院 | 由il－15刺激和活化毛发生长的方法 |
| CN108147990A (zh) * | 2016-12-02 | 2018-06-12 | 上海中医药大学 | 一种膜锚定元件及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| Human IL-15 variant T95X,SEQ ID 8, XP002807948.DATABASE Geneseq ebi.2014, * |
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| JP2022550743A (ja) | 2022-12-05 |
| GB2602733A (en) | 2022-07-13 |
| CN112552391A (zh) | 2021-03-26 |
| US20220348624A1 (en) | 2022-11-03 |
| EP4015526A4 (en) | 2023-01-04 |
| AU2020354198A1 (en) | 2022-03-24 |
| CA3153542A1 (en) | 2021-04-01 |
| GB202202934D0 (en) | 2022-04-20 |
| WO2021057826A1 (zh) | 2021-04-01 |
| EP4015526A1 (en) | 2022-06-22 |
| KR20220069018A (ko) | 2022-05-26 |
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