CN117264958A - S100a9适配体及其筛选方法和应用 - Google Patents
S100a9适配体及其筛选方法和应用 Download PDFInfo
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Abstract
本发明涉及S100A9适配体及其筛选方法和应用。本发明通过SELEX筛选得到了特异性结合和/或抑制S100A9的适配体。该S100A9适配体,能够特异性结合S100A9,因此可用于从复杂体系中捕获S100A9,适用于S100A9蛋白的纯化、检测、体内成像、药物递送及S100A9相关疾病的诊断,其同时还能够抑制S100A9的功能,因此同样适用于S100A9相关疾病的治疗,包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。本发明的优选S100A9适配体可以抑制S100A9介导的滑膜成纤维细胞的激活,适用于类风湿性关节炎的治疗。
Description
技术领域
本发明涉及生物医药领域,更具体地说,涉及S100A9适配体及其筛选方法和应用。
背景技术
S100家族是一类结构和功能均具有相似性的低分子钙结合蛋白。S100A9是S100家族中的重要成员,也被称为髓系相关蛋白14(MRP14),在体内不同条件下可形成同源二聚体、异源二聚体、四聚体,通常以S100A8/S100A9异源二聚体(即钙卫蛋白)形式存在,在细胞内参与调节微管重组、整合丝裂原活化蛋白激酶活性、花生四烯酸代谢和运输、钙依赖性信号等。在细胞外,S100A9从活化或坏死的中性粒细胞、单核细胞和树突细胞等免疫细胞中以组成型表达,约占中性粒细胞溶质蛋白的45%、单核细胞的1%,以颗粒形式储存,响应炎症、感染性刺激而释放,作为先天免疫介质参与各种炎性疾病的发生,放大先天免疫反应促进炎症。另外,S100A9也在多种肿瘤中表达升高,参与肿瘤细胞的生长、迁移、侵袭、凋亡和耐药等进程。目前,S100A9已被认为是炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤等的潜在生物标志物和治疗靶点。
适配体(aptamer)通常是指在体外利用指数富集的配基系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)筛选得到的寡核苷酸序列(ssDNA或RNA),它能与靶标高亲和力、高特异性的结合。适配体通过分子内碱基配对形成如假结、发夹、凸环、G-四链体等稳定的二级结构,从而使其具有特定的分子构象。适配体与抗体相似,被称为“化学抗体”。但与传统抗体相比,适配体有着更大的靶标范围,包括氨基酸、金属离子等小分子物质,以及生长因子、酶等生物大分子,甚至完整的细菌和细胞等。此外,适配体还具有独特的优势,包括尺寸小、成本低、稳定性高和免疫原性低等。基于这些特点,适配体被广泛用于生物标志物发现、细胞识别和分选、生物传感、生物成像、疾病诊断和药物靶向递送等。除此以外,适配体自身也可作为新型药物,直接用于疾病治疗,减少毒副作用。然而,目前缺乏特异性结合和/或抑制S100A9的适配体。
发明内容
因此,本发明针对目前缺乏特异性结合和/或抑制S100A9的适配体的技术问题,提供一种S100A9适配体,其能够特异性结合S100A9,因此可用于从复杂体系中捕获S100A9,适用于S100A9蛋白的纯化、检测、体内成像、药物递送及S100A9相关疾病的诊断,其同时还能够抑制S100A9的功能,因此同样适用于S100A9相关疾病的治疗。
本发明的第一目的在于,提供一种S100A9适配体,所述S100A9适配体包含以下序列中的至少一种:
(1)SEQ ID NO.1所示序列或其衍生物;
(2)SEQ ID NO.2所示序列或其衍生物;
SEQ ID NO.1:5’-GTTGCAACTGGGCTGACCGTGTTAAACCCG-3’;
SEQ ID NO.2:5’-ACCTGCACTTAGAGGGCATTAAACGCTGGG-3’;
所述S100A9适配体为特异性结合S100A9的适配体、抑制S100A9的适配体、或同时特异性结合和/或抑制S100A9的适配体。
在本发明的优选实施例中,所述SEQ ID NO.1所示序列的衍生物包括:所述SEQ IDNO.1所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ ID NO.1所示序列的同源性在60%以上的序列、与所述SEQ ID NO.1所示序列进行杂交的序列、由所述SEQ ID NO.1所示序列进行转录的序列、所述SEQ ID NO.1所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.1所示序列改造成的肽核酸;
所述SEQ ID NO.2所示序列的衍生物包括:所述SEQ ID NO.2所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ ID NO.2所示序列的同源性在60%以上的序列、与所述SEQ ID NO.2所示序列进行杂交的序列、由所述SEQ ID NO.2所示序列进行转录的序列、所述SEQ ID NO.2所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.2所示序列改造成的肽核酸。
硫代磷酸酯修饰是最简单和广泛使用的可应用于增加核酸酶抗性的化学修饰,未经修饰的核酸适配体可以显示活性,但他们会被核酸酶迅速降解,因此作用有限。在寡核苷酸的磷酸酯骨架上,硫代磷酸酯键用硫原子取代了非桥键氧原子,使得核苷酸间键抵抗核酸酶的降解从而更稳定。肽核酸(peptide nucleic acids,PNA)是一类以多肽骨架取代糖磷酸主链的DNA类似物,以中性的肽链酰胺2氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余的与DNA相同。PNA可以通过Watson Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构,有很高的杂交稳定性、优良的特异序列识别能力、不被核酸酶和蛋白酶水解,并可以与配基相连共转染进入细胞。因此,所述SEQ ID NO.1所示序列的衍生物优选为所述SEQ ID NO.1所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQID NO.1所示序列改造成的肽核酸;所述SEQ ID NO.2所示序列的衍生物优选为所述SEQ IDNO.2所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.2所示序列改造成的肽核酸。本领域技术人员可以采用本领域的常规方法制备前述硫代磷酸酯骨架序列和肽核酸,在此不再累述。
优选地,所述至少一个碱基被修饰包括磷酸化、甲基化、氨基化、巯基化或同位素化,或被替换成锁核酸(locked nucleic acid,LNA)、非锁核酸(unlocked nucleic acid,UNA)或者乙二醇核酸(Glycol nucleic acid,GNA)、吗啉核酸(Morpholine nucleic acid,MNA)、肽核酸(Peptide nucleic acid,PNA)、苏阿糖核酸(Sua nucleic acid,SNA)或桥联核酸(Bridged nucleic acid,BNA)。
优选地,可在所述SEQ ID NO.1所示序列或其衍生物或所述SEQ ID NO.2所示序列或其衍生物的3’端、5’端或中间碱基接功能基团。所述功能基团包括荧光基团、放射性基团、治疗性药物、生物素、地高辛、纳米发光材料、核酸物质或酶标记物中的至少一种。
在本发明中,可以采用任何已知方法,采用任何已知基团对SEQ ID NO.1~2所示序列进行修饰和功能基团连接,只要其不影响所获序列的性能即可,即对SEQ ID NO:1~2所示序列进行修饰或功能基团连接获得的衍生物并不会丧失其基本功能,即特异性结合和/或抑制S100A9的适配体。这些修饰或者功能基团可以用于提高适配体的稳定性、提供检测信号,或者用于连接适配体与其他物质形成组合物。
本发明的所述S100A9适配体可以特异性识别S100A9,从而从复杂体系中捕获S100A9,因此可用于S100A9的捕获、检测、纯化、体内成像和诊断、药物递送等。
本发明的所述S100A9适配体还可以抑制S100A9的功能,从而用于S100A9相关疾病的治疗。优选地,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。
本发明的第二目的在于,提供本发明的第一方面的所述S100A9适配体在制备检测S100A9的产品、纯化S100A9的产品、体内成像S100A9的产品、诊断S100A9相关疾病的产品、治疗S100A9相关疾病的产品、或递送药物至S100A9相关疾病部位的产品中的应用。
如前所述,在本领域中,适配体被广泛用于生物标志物发现、细胞识别和分选、生物传感、生物成像、疾病诊断和药物靶向递送等。因此,在本领域技术人员获得本发明的前述S100A9适配体,其可以基于本领域中的公知常识或者惯用技术手段,将前述S100A9适配体应用于制备检测S100A9的产品、纯化S100A9的产品、体内成像S100A9的产品、诊断S100A9相关疾病的产品、治疗S100A9相关疾病的产品、或递送药物至S100A9相关疾病部位的产品。
本发明的第三目的在于,提供本发明的第一方面的所述S100A9适配体在制备S100A9相关疾病的药物中的应用,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。
如前所述,由于S100A9已被认为是炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤等的潜在生物标志物和治疗靶点。因此在本领域技术人员获得本发明的前述S100A9适配体,其可以基于本领域中的公知常识或者惯用技术手段,将前述S100A9适配体应用于制备所述S100A9相关疾病的药物。
在本发明的优选实施例中,所述S100A9适配体在类风湿性关节炎中有很好的治疗效果。因此,所述S100A9适配体优选适用于制备治疗类风湿性关节炎的药物。进一步地,所述S100A9适配体优选与阿达木单抗一起用于制备治疗类风湿性关节炎的药物。
本发明的第四目的在于,提供一种试剂盒或检测芯片,包括本发明的第一方面的所述S100A9适配体,所述试剂盒或检测芯片用于检测S100A9、纯化S100A9、体内成像S100A9、诊断S100A9相关疾病、治疗S100A9相关疾病或递送药物至S100A9相关疾病部位。
如前所述,适配体被广泛用于生物标志物发现、细胞识别和分选、生物传感、生物成像、疾病诊断和药物靶向递送等。同时S100A9已被认为是炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤等的潜在生物标志物和治疗靶点。因此,在本领域技术人员获得本发明的前述S100A9适配体,其可以基于本领域中的公知常识或者惯用技术手段,制备前述试剂盒或检测芯片。
本发明的第五目的在于,提供一种药物组合,包括所述S100A9适配体;所述药物组合物用于治疗S100A9相关疾病,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。
在本发明的优选实施例中,所述药物组合物包括用于治疗类风湿性关节炎的药物组合物,所述药物组合物进一步包括疾病调节抗风湿药物、生物类疾病调节抗风湿药物、靶向合成疾病调节抗风湿药物、非甾体类抗炎药、免疫抑制剂或糖皮质激素。
优选地,所述疾病调节抗风湿药物包括甲氨蝶呤、来氟米特、羟氯喹和柳氮磺吡啶。优选地,所述生物类疾病调节抗风湿药物包括阿巴西普、阿达木单抗、阿那白滞素、培塞利珠单抗、依那西普、戈利木单抗、英夫利西单抗、利妥昔单抗、沙利鲁单抗和托珠单抗。优选地,所述靶向合成疾病调节抗风湿药物包括巴瑞替尼、托法替布和乌帕替尼。优选地,所述非甾体类抗炎药包括布洛芬、双氯芬酸、吲哚美辛、美洛昔康、塞来昔布。优选地,所述免疫抑制剂包括环丙酰胺、硫唑嘌呤、霉酚酸酯、环孢素。优选地,所述糖皮质激素包括强的松、泼尼松、曲安奈德。
在本发明的进一步的优选实施例中,所述药物组合物包括所述S100A9适配体和阿达木单抗。所述S100A9适配体与阿达木单抗联合用药在类风湿性关节炎中可以达到很好的治疗效果。
本发明的第六目的在于,提供一种S100A9适配体的筛选方法,包括以下步骤:
S1、合成初始随机单链DNA文库、正向引物和反向引物,所述文库包括如SEQ IDNO.3所示的随机单链DNA序列,所述正向引物包括如SEQ ID NO.4所示序列,所述反向引物包括如SEQ ID NO.5所示序列;SEQ ID NO.3:5’-TGAGAATATGTAGACGATCC-(N)-CGGAGCTTCAAGATGATCTG-3’;SEQ ID NO.4:5’-TGAGAATATGTAGACGATCC-3’;SEQ ID NO.5:5’-CAGATCATCTTGAAGCTCCG-3’;其中N表示20~60个随机DNA寡核苷酸;
S2、选取S100A9为靶蛋白,固定在蛋白纯化磁珠上,用于正向筛选,并选取未固定S100A9的蛋白纯化磁珠为对照,用于负向筛选;
S3、将所述文库和所述S100A9进行孵育后洗涤,然后洗脱与S100A9结合的序列并取第一上清;
S4、对所述第一上清中富集的单链序列进行扩增和分离以获得富集单链文库;
S5、将步骤S4中获得的富集单链文库重复所述步骤S3和S4至少三轮之后,采用所述未固定S100A9的蛋白纯化磁珠作为对照与所述富集单链文库进行孵育后取第二上清;
S6、将所述步骤S5中的第二上清用于顺序执行步骤S3和S4之后,按照步骤S5,步骤S3和步骤S4的顺序重复执行所述步骤S5、步骤S3和步骤S4;直至选出与S100A9结合能力达到峰值且与所述对照磁珠无结合的富集文库并进行测序以获得所述S100A9适配体。
在本发明的优选实施例中,所述S100A9适配体包含以下序列中的至少一种:
(1)SEQ ID NO.1所示序列或其衍生物;
(2)SEQ ID NO.2所示序列或其衍生物;
SEQ ID NO.1:5’-GTTGCAACTGGGCTGACCGTGTTAAACCCG-3’;
SEQ ID NO.2:5’-ACCTGCACTTAGAGGGCATTAAACGCTGGG-3’;
所述S100A9适配体为特异性结合S100A9的适配体、抑制S100A9的适配体、或同时特异性结合和/或抑制S100A9的适配体。
在本发明的优选实施例中,所述SEQ ID NO.1所示序列的衍生物包括:所述SEQ IDNO.1所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ ID NO.1所示序列的同源性在60%以上的序列、与所述SEQ ID NO.1所示序列进行杂交的序列、由所述SEQ ID NO.1所示序列进行转录的序列、所述SEQ ID NO.1所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.1所示序列改造成的肽核酸;
所述SEQ ID NO.2所示序列的衍生物包括:所述SEQ ID NO.2所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ ID NO.2所示序列的同源性在60%以上的序列、与所述SEQ ID NO.2所示序列进行杂交的序列、由所述SEQ ID NO.2所示序列进行转录的序列、所述SEQ ID NO.2所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.2所示序列改造成的肽核酸。
优选地,所述至少一个碱基被修饰包括磷酸化、甲基化、氨基化、巯基化或同位素化,或被替换成锁核酸、非锁核酸或者乙二醇核酸、吗啉核酸、肽核酸、苏阿糖核酸或桥联核酸。优选地,可在所述SEQ ID NO.1所示序列或其衍生物或所述SEQ ID NO.2所示序列或其衍生物的3’端、5’端或中间碱基接功能基团。所述功能基团包括荧光基团、放射性基团、治疗性药物、生物素、地高辛、纳米发光材料、核酸物质或酶标记物中的至少一种。
在本发明的优选实施例中,在所述步骤S1中,N表示30个随机DNA寡核苷酸。
在本发明的优选实施例中,在所述步骤S2中,所述S100A9为His标签的S100A9、Myc标签的S100A9、HA标签的S100A9、GST标签的S100A9、或无标签的S100A9,优选为His标签的S100A9,所述对照磁珠优选为未固定S100A9的His标签蛋白纯化磁珠。
在本发明的优选实施例中,在所述步骤S3中,取200pmol~10nmol初始随机单链DNA文库溶解于结合缓冲液(优选为包含5mM MgCl2的磷酸盐缓冲盐溶液(PBS))中,80~100℃加热3~10分钟后放置于冰上5~10分钟,进行正向筛选;即将溶于所述结合缓冲液的初始随机单链DNA文库与固定在His标签蛋白纯化磁珠表面的50~1000pmol His标签的S100A9蛋白在4~37℃孵育0.5~3小时。孵育结束后将磁珠用洗涤缓冲液(优选为包含5mMMgCl2,5mM咪唑,0.02%吐温20的PBS)洗3~5次。磁珠中加入5~50μL 50~500mM咪唑,室温静置1~10分钟,将磁珠放在磁力架上,待磁液分离后,取第一上清。
在本发明的优选实施例中,在所述步骤S4中,可以采用链霉亲和素磁珠法、不对称PCR、酶消化法、电泳分离法及这些方法的组合分离从所述第一上清中扩增的富集单链文库。
在本发明的进一步的优选实施例中,在所述步骤S4中,采用所述正向引物和5’端生物素标记的所述反向引物对所述第一上清进行PCR扩增,优选先取部分所述第一上清用循环数梯度实验确定扩增最适的循环数,再挑选最适循环数对剩余上清进行PCR扩增;将扩增后的双链DNA产物与链霉亲和素磁珠在常温下孵育0.1~1小时,双链DNA产物上存在的生物素与链霉亲和素磁珠结合,从而固定在磁珠表面;然后将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3~5次;为了分离下一轮筛选用的单链序列,在磁珠中加入50~200mM NaOH溶液,反应5~30分钟,将磁珠放在磁力架上,待磁液分离后,带生物素的一条DNA单链留在链霉亲和素磁珠上,不带生物素的一条DNA单链留在获得的上清中,收集所述上清,使用1M乙酸溶液中和,加入脱盐柱中进行脱盐,获得所述富集单链文库。
在本发明的进一步的优选实施例中,在所述步骤S5中,将获得的所述富集单链文库(即其中的每个单链序列)重复上述步骤S3和S4至少三轮之后,引入负向筛选,即取200pmol~10nmol所述富集单链文库溶解于所述结合缓冲液中,80~100℃加热3~10分钟后放置于冰上5~10分钟,然后将所述富集单链文库与未固定S100A9的His标签蛋白纯化磁珠于4~37℃孵育0.5~3小时,孵育结束后离心收集所述第二上清。
在本发明的进一步的优选实施例中,在所述步骤S6中,通过多轮筛选特异性结合S100A9的序列,在筛选过程中对不同筛选轮数的序列与所述His标签的S100A9的结合情况进行检测,直至所述富集序列对所述S100A9达到峰值且与所述对照无结合,停止所述筛选,进行测序以获得所述S100A9适配体
本发明的所述S100A9适配体,其能够特异性结合S100A9,因此可用于从复杂体系中捕获S100A9,适用于S100A9蛋白的纯化、检测、体内成像、药物递送及S100A9相关疾病的诊断,其同时还能够抑制S100A9的功能,因此同样适用于S100A9相关疾病的治疗。因此,本发明所述S100A9适配体可以用于制备检测S100A9的产品、纯化S100A9的产品、体内成像S100A9的产品、诊断S100A9相关疾病的产品、治疗S100A9相关疾病的产品、或递送药物至S100A9相关疾病部位的产品,用于制备S100A9相关疾病的药物,其中所述产品包括试剂盒或检测芯片,所述药物可进一步包括任何可与所述S100A9适配体进行联合用药的第二药物,例如疾病调节抗风湿药物、生物类疾病调节抗风湿药物、靶向合成疾病调节抗风湿药物、非甾体类抗炎药、免疫抑制剂或糖皮质激素。
基于以上公开的内容,本领域技术人员知悉,所述S100A9适配体能够特异性结合S100A9,因此可用于从复杂体系中捕获S100A9,适用于S100A9蛋白的纯化、检测、体内成像、药物递送及S100A9相关疾病的诊断,其同时还能够抑制S100A9的功能,因此同样适用于S100A9相关疾病的治疗。
附图说明
下面将结合附图及实施例对本发明作进一步说明,附图中:
图1为SELEX筛选特异性结合和/或抑制S100A9的适配体的流程;
图2A-2B为不同筛选轮数富集到的序列与S100A9和对照磁珠的结合;
图3A-3E为最优适配体SA9-03和SA9-05与S100A9和对照磁珠的结合及二级结构;
图4A-4P为SA9-03和SA9-05对S100A9介导的FLS激活的抑制效果;
图5A-5E为SA9-03和SA9-05在CIA小鼠模型中的治疗效果。
具体实施方式
在本发明的优选实施例中,通过SELEX筛选得到了特异性结合和/或抑制S100A9的适配体SA9-03和SA9-05(即SEQ ID NO.1所示序列和SEQ ID NO.2所示序列),筛选过程中选取His标签的S100A9为靶蛋白,固定在His标签蛋白纯化磁珠上,用于正向筛选。选取未固定S100A9的His标签蛋白纯化磁珠为对照,用于负向筛选。随着SELEX筛选的进行,富集文库与S100A9的结合有显著增多且不结合对照磁珠。筛选完成后测序得到的最优适配体SA9-03和SA9-05浓度依赖性结合S100A9且不与对照磁珠结合。
炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤中都有S100A9的高表达,且S100A9促进了这些疾病的发生和发展。S100A9主要与多种细胞表面的TLR4、RAGE、CD33、CD68、CD69或CD147结合,激活下游疾病相关信号通路。因此,SA9-03和SA9-05的给药可以有效抑制S100A9介导的TLR4、RAGE、CD33、CD68、CD69或CD147下游信号通路的激活,从而用于与S100A9相关的炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤的治疗。
进一步地,在类风湿性关节炎中,S100A9作用于滑膜成纤维细胞(FLS),提高趋化因子、炎症因子和抗原提呈相关基因表达,促进FLS增殖、侵袭、迁移和凋亡抵抗,最终导致滑膜成纤维细胞激活,加重关节炎的发病。SA9-03和SA9-05可以抑制S100A9介导的FLS的激活。胶原诱导的关节炎(collagen-induced arthritis,CIA)小鼠是常用的模拟人类风湿性关节炎动物模型。S100A9在CIA小鼠中水平升高且促进关节炎。SA9-03和SA9-05全身给药可以抑制CIA小鼠模型的疾病进展。SA9-03或SA9-05与TNF单抗阿达木单抗(Adalimumab)联合给药可以更有效的抑制CIA小鼠疾病进展。为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均从常规生化试剂公司购买得到。
实施例一、SELEX筛选特异性结合和/或抑制S100A9的适配体
SELEX筛选特异性结合和/或抑制S100A9适配体的流程,如图1所示,包括以下步骤。
(1)合成初始随机单链DNA文库,正向引物和反向引物:文库序列如SEQ ID NO.3所示,SEQ ID NO.3:5’-TGAGAATATGTAGACGATCC-(30N)-CGGAGCTTCAAGATGATCTG-3’;其中30N为30个随机DNA寡核苷酸。正向引物如SEQ ID NO.4所示,SEQ ID NO.4:5’-TGAGAATATGTAGACGATCC-3’;反向引物如SEQ ID NO.5所示,SEQ ID NO.5:5’-CAGATCATCTTGAAGCTCCG-3’。文库、正向引物和反向引物均由上海生工生物有限公司合成。
(2)确定靶蛋白和对照:选取His标签的S100A9为靶蛋白,固定在His标签蛋白纯化磁珠上,用于正向筛选。选取未固定S100A9的His标签蛋白纯化磁珠为对照,用于负向筛选。His标签(His-tag)又称多组氨酸标签,由6到10个连续的组氨酸残基组成。最常用的His标签是6xHis标签,其分子量为0.8kDa。该标签用于许多重组蛋白,可帮助蛋白纯化,使研究人员能够从细胞或细胞裂解液中的数千个蛋白中提取靶蛋白。当然,在本发明的其他优选实施例中,也可以直接使用S100A9为靶蛋白。
(3)正向筛选:350μL含有250pmol His标签的S100A9蛋白溶液中加入50μL His标签蛋白纯化磁珠,室温孵育30分钟,使得His标签的S100A9固定在磁珠表面。取2nmol初始单链DNA文库溶解于500μL结合缓冲液(PBS,包含5mM MgCl2)中,95℃加热5分钟后放置于冰上10分钟,加入至上述磁珠中常温孵育1小时。磁珠用洗涤缓冲液(PBS,包含5mM MgCl2,5mM咪唑,0.02%吐温20)洗3次,每次1mL。磁珠中加入50μL 250mM咪唑,室温静置1分钟,将磁珠放在磁力架上,待磁液分离后,取上清。
(4)磁珠法分离富集的单链序列:使用上述正向引物和5’端生物素标记的反向引物对收集的上清进行PCR扩增。先取部分上清用循环数梯度实验确定扩增最适的循环数,再挑选最适循环数对剩余上清进行PCR扩增。扩增后的双链DNA产物与链霉亲和素磁珠在室温孵育30分钟,双链DNA产物上的生物素与链霉亲和素磁珠结合,从而固定在磁珠表面。将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3次。为了分离下一轮筛选用的单链序列,在磁珠中加入500μL 100mM NaOH溶液,反应15分钟,将磁珠放在磁力架上,待磁液分离后,带生物素的一条DNA单链留在链霉亲和素磁珠上,不带生物素的一条DNA单链留在上清中。收集上清,使用1M乙酸溶液中和,加入脱盐柱中进行脱盐,获得富集单链序列。
(5)引入负向筛选:将所述富集单链序列重复上述步骤(3)和(4)。待重复至第三轮及以后,引入负向筛选。在负向筛选中,取2nmol富集单链文库溶解于450μL结合缓冲液中,95℃加热5分钟后放置于冰上10分钟。将富集文库与50μL未固定S100A9的His标签蛋白纯化磁珠室温孵育30分钟。将磁珠放在磁力架上,待磁液分离后,收集上清。
(6)多轮筛选特异性结合S100A9的序列:对步骤(5)收集的上清继续进行上述步骤(3)和(4)的操作。然后再依次重复步骤(5)、(3)和(4)的操作多轮。重复筛选过程中富集序列与S100A9的孵育时间逐渐缩短至30分钟,洗涤次数逐渐提高至5次,确保富集到的序列与S100A9有较强的结合能力。筛选过程中检测不同筛选轮数的序列与S100A9的结合,直至富集的序列对S100A9的结合不再增多且与对照磁珠无明显结合后即可停止。
具体如下:将250pmol不同筛选轮数(0、4、6、8、10轮)的富集文库与固定于磁珠的500pmol His标签的S100A9或未固定S100A9的对照磁珠于室温孵育1小时。将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3次。洗脱与S100A9或对照磁珠结合的序列并浓缩后使用超微量分光光度计(NanoDrop)定量。结果如图2A所示。该结果表明随着筛选轮数的进行,富集序列与S100A9的结合逐渐增多且与对照磁珠无明显结合。
使用反向引物和5’端生物素标记的正向引物对不同筛选轮数(0、4、6、8、10轮)的富集序列分别进行PCR扩增。扩增后的双链DNA产物与链霉亲和素磁珠在室温孵育30分钟,双链DNA产物上的生物素与链霉亲和素磁珠结合,从而固定在磁珠表面。将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3次。在磁珠中加入500μL 100mM NaOH溶液,反应15分钟,将磁珠放在磁力架上,待磁液分离后,带生物素的一条DNA单链留在链霉亲和素磁珠上,不带生物素的一条DNA单链留在上清中。移除上清,用PBS洗磁珠3次。洗脱磁珠上的生物素标记的不同筛选轮数(0、4、6、8、10轮)的富集序列。将500pmol S100A9蛋白与固定于链霉亲和素磁珠的250pmol生物素标记的不同筛选轮数(0、4、6、8、10轮)的富集序列或对照磁珠于室温孵育1小时。将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3次。洗脱与富集文库或对照磁珠结合的S100A9并浓缩后使用超微量分光光度计(NanoDrop)定量。结果如图2B所示。该结果表明随着筛选轮数的进行,富集序列与S100A9的结合逐渐增多且与对照磁珠无明显结合。
本领域技术人员知悉,SELEX筛选被广泛应用于适配体的筛选中。因此,在本发明的其他优选实施例中,本领域技术人员可以根据实际情况,对筛选过程中采用的试剂,操作步骤进行调整,从而获得本发明的S100A9适配体,这些都落入本发明的保护。进一步地,与其他可能的实现方式相比,采用本发明的实施例中的试剂和步骤,筛选过程更加简便、快速、经济,并且分辨率更高,亲和力更强。
实施例二、最优适配体与S100A9和对照磁珠的结合及二级结构
(1)上述SELEX筛选最后富集到的单链DNA序列经克隆测序,挑选富集程度最高的序列(SA9-03和SA9-05)。将不同浓度(0、100、200、300、400、500pmol)SA9-03、SA9-05或阴性对照(negative control,NC)与固定于磁珠的500pmol His标签的S100A9或对照磁珠于室温孵育1小时。将磁珠放在磁力架上,待磁液分离后,移除上清,用PBS洗磁珠3次。洗脱与S100A9或对照磁珠结合的序列并浓缩后使用超微量分光光度计(NanoDrop)定量。结果如图3A-3B所示。该结果表明随着浓度的增加,SA9-03和SA9-05与S100A9的结合能力逐渐提高且与对照磁珠无明显结合,SA9-05的效果好于SA9-03。SA9-03序列如SEQ ID NO.1所示,SEQID NO.1:5’-GTTGCAACTGGGCTGACCGTGTTAAACCCG-3’;SA9-05序列如SEQ ID NO.2所示,SEQID NO.2:5’-ACCTGCACTTAGAGGGCATTAAACGCTGGG-3’;NC序列如SEQ ID NO.6所示,SEQ IDNO.6:5’-AAACGGTGGCTTACCGTCCGAAAGCTTTCC-3’。使用RNAstructure软件预测SA9-03和SA9-05的二级结构,如图3C所示。
(2)斑点杂交(Dot Blot)是分子生物学中用于检测、分析和鉴定蛋白质/核酸的一种技术。实验原理是将蛋白点到硝酸纤维素膜/醋酸纤维素膜固相支持物上,随后用已标记的探针(核酸或蛋白)进行杂交和放射自显影,判断是否有杂交及其杂交强度,进行两者结合能力的验证,结合能力与显影光强呈正比。在本实施例中,将硝酸纤维素膜剪成小圆片,放入96孔板底部。实验孔加入1μg S100A9蛋白,对照孔加入超纯水,晾干后用TBST缓冲液洗3次。用5%脱脂牛奶封闭60分钟,TBST缓冲液洗3次。加入100μL 10μM生物素标记的NC、SA9-03和SA9-05,室温孵育2小时,用TBST缓冲液洗3次。再加入辣根过氧化物酶标记的链霉亲和素,室温孵育1小时。最后使用ECL化学发光检测试剂盒进行显影。与NC序列相比,SA9-03和SA9-05均可以与S100A9蛋白结合,如图3D所示。
(3)药物亲和反应靶点稳定技术(drug affinity responsive targetstability,DARTS)是一种新型靶标鉴定方法,它的原理是利用药物分子与靶蛋白结合后,形成分子与蛋白的复合物,进而增加靶蛋白的稳定性,使其具有抵抗酶解的特性。在含有100μg S100A9蛋白的20μL反应体系中加入PBS或1000nM NC、SA9-03或SA9-05,混匀后室温孵育1小时,再加入终浓度0.03%的链霉蛋白酶(Pronase),混匀后室温孵育30分钟。随后进行蛋白免疫印迹(western blot)实验。结果表明,与NC相比,SA9-03和SA9-05均可以抵抗蛋白酶降解,其中SA9-05的效果好于SA9-03。结果如图3E所示。
基于以上实验可知,SA9-03和SA9-05可以特异性识别S100A9,从而从复杂体系中捕获S100A9,因此可用于S100A9的检测、纯化、体内成像和诊断、药物递送等。
例如可以采用荧光标记S100A9适配体,在S100A9适配体与S100A9结合后,采用流式细胞仪、荧光分光光度计可以检测S100A9适配体复合物,从而进行S100A9的检测。采用荧光显微镜能够呈现结合物的直观图像,从而实现体内成像和诊断。又例如,可以在S100A9适配体的末端修饰不同的化学基团,使之与药物载体连接,制备出不同功能的药物传递系统,例如S100A9适配体可以和聚合物、无机纳米粒子、树枝状分子、脂质体、胶束连接形成不同的药物传递系统。另外,S100A9适配体也可连接多种标记物,应用于样本细胞、组织或生物大分子的检测与鉴定。也可以将标记物标记于S100A9适配体的5′端和/或3′后导入待检测样品,可使用多种手段检测适配体与待测样品的结合情况,例如采用流式细胞仪,共聚焦显微镜检测标记了荧光的核酸适配体,采用免疫化学发光检测方法检测标记了地高辛的适配体,从而适用于多种检测方法和系统。并且,将药物分子嵌入S100A9适配体是一种简单而有效的靶向递药方式,药物也可经化学修饰形成稳定的酯、胺和二硫键结合至适配体上或通过连接子共价结合。
因此,SA9-03和SA9-05可用于制备检测S100A9的产品、纯化S100A9的产品、体内成像S100A9的产品、诊断S100A9相关疾病的产品、治疗S100A9相关疾病的产品、或递送药物至S100A9相关疾病部位的产品中的应用,尤其采用其构建试剂盒或者检测芯片。
进一步地,本领域技术人员知悉,可以在不影响所述SA9-03和SA9-05序列的基本功能,即特异性结合和/或抑制S100A9的基础上,对其进行改造,修饰,从而获得对应的衍生物。例如所述SA9-03序列的衍生物包括:所述SA9-03序列的至少一个碱基被修饰获得的衍生物、与所述SA9-03序列的同源性在60%以上的序列、与所述SA9-03序列进行杂交的序列、由所述SA9-03序列进行转录的序列、所述SA9-03序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-03序列改造成的肽核酸;所述SA9-05序列的衍生物包括:所述SA9-05序列的至少一个碱基被修饰获得的衍生物、与所述SA9-05序列的同源性在60%以上的序列、与所述SA9-05序列进行杂交的序列、由所述SA9-05序列进行转录的序列、所述SA9-05序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-05序列改造成的肽核酸。
我们采用所述SA9-03序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-03序列改造成的肽核酸、所述SA9-05序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-05序列改造成的肽核酸重复上述实验,其结果证明,这些SA9-03序列的衍生物和SA9-05序列的衍生物同样能够与S100A9蛋白结合并抵抗蛋白酶降解。
因此,如前所述,SA9-03序列的衍生物和SA9-05序列的衍生物同样可以特异性识别S100A9,从而从复杂体系中捕获S100A9,因此可用于S100A9的检测、纯化、体内成像和诊断、药物递送等。
实施例三、SA9-03和SA9-05对S100A9介导的FLS激活的抑制效果
滑膜成纤维细胞(fibroblast-like synoviocytes,FLS)是一种间充质细胞,主要位于滑膜内层。正常生理情况下,FLS与关节稳态有关,为软骨提供细胞外基质。然而,在慢性自身免疫病类风湿性关节炎发病过程中,FLS是增生性滑膜的核心效应细胞。该细胞在炎性微环境刺激下发生激活并分化为侵袭性亚型,产生细胞因子、趋化因子和基质降解分子,并迁移至关节软骨处侵蚀破坏软骨和骨结构。异常的FLS具有许多肿瘤样病变,如摆脱生长限制,获得侵袭、迁移和抗凋亡等能力。在类风湿性关节炎中,S100A9作用于滑膜成纤维细胞(FLS),提高趋化因子、炎症因子和抗原提呈相关基因表达,促进FLS增殖、侵袭、迁移和凋亡抵抗,最终导致FLS的激活,加重关节炎的发病。为了验证SA9-03和SA9-05是否可以抑制S100A9介导的FLS激活,具体实验如下:
(1)在类风湿性关节炎病人来源的FLS中加入5μg/mL S100A9和不同浓度(0、100、250、500和1000nM)SA9-03、SA9-05或NC序列,37℃环境下孵育30小时。实时定量PCR检测趋化因子(CXCL11)、炎症因子(IL-6和IL-1β)和抗原提呈相关基因(HLA-DRB1)的水平,其结果如图4A-4L所示。
针对趋化因子(CXCL11),参见图4A可知,在添加5μg/mL S100A9时,趋化因子的相对表达水平明显升高。因此可知,S100A9作用于滑膜成纤维细胞(FLS),可以显著提高趋化因子的相对表达水平。在添加5μg/mL S100A9+100、250、500或1000nMNC序列时,趋化因子的相对表达水平变化不大。因此可知,NC序列对趋化因子的相对表达水平变化并无显著影响。而参见图4E可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-03序列时,趋化因子的相对表达水平明显浓度依赖地降低。同样地,参见图4I可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-05序列时,趋化因子的相对表达水平也明显浓度依赖地降低。
针对炎症因子(IL-6和IL-1β),参见图4B和图4C可知,在添加5μg/mL S100A9时,炎症因子相对表达水平明显升高。因此可知,S100A9作用于滑膜成纤维细胞(FLS),可以显著提高炎症因子(IL-6和IL-1β)的相对表达水平。在添加5μg/mL S100A9+100、250、500或1000nMNC序列时,炎症因子的相对表达水平变化不大。因此可知,NC序列对炎症因子的相对表达水平变化并无显著影响。而参见图4F和图4G可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-03序列时,炎症因子的相对表达水平明显浓度依赖地降低。同样地,参见图4J和图4K可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-05序列时,炎症因子的相对表达水平也明显浓度依赖地降低。
针对抗原提呈相关基因(HLA-DRB1),参见图4D可知,在添加5μg/mL S100A9时,抗原提呈相关基因的相对表达水平明显升高。因此可知,S100A9作用于滑膜成纤维细胞(FLS),可以显著提高抗原提呈相关基因的相对表达水平。而在添加5μg/mL S100A9+100、250、500或1000nMNC序列时,抗原提呈相关基因的相对表达水平变化不大。因此可知,NC序列对抗原提呈相关基因的相对表达水平变化并无显著影响。而参见图4H可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-03序列时,抗原提呈相关基因的相对表达水平明显浓度依赖地降低。同样地,参见图4L可知,在加入5μg/mL S100A9+100、250、500或1000nMSA9-05序列时,抗原提呈相关基因的相对表达水平也明显浓度依赖地降低。
综上所述,前述结果表明与NC对比,SA9-03和SA9-05均可以浓度依赖性抑制S100A9介导的FLS的趋化因子、炎症因子和抗原提呈相关基因的相对表达水平,其中SA9-05的效果好于SA9-03,并且浓度越大,趋化因子、炎症因子和抗原提呈相关基因的相对表达水平越低。
(2)在FLS中加入1000nM SA9-03、SA9-05或NC序列和5μg/mL S100A9,37℃环境下孵育3天,进行细胞计数CCK-8(Cell Counting Kit-8)实验。实验过程中每天更换含有1000nM SA9-03、SA9-05或NC序列和5μg/mL S100A9的培养基。用酶标仪检测450nm处的吸光度值,其结果如图4M所示。该结果表明,与NC对比,SA9-03和SA9-05均可以抑制S100A9介导的FLS的细胞增殖,SA9-05的效果好于SA9-03。
(3)在FLS中加入5μg/mL S100A9和1000nMSA9-03、SA9-05或NC序列,37℃环境下孵育30小时,进行侵袭(Transwell)和迁移实验。进行侵袭实验时,将实验所用到的聚碳酸酯膜侵袭上室预冷处理,用无血清的培养基稀释基底膜基质(Matrigel)胶,加入到上室。将铺好的胶放于培养箱中,使其充分凝固。用无血清的培养基稀释经S100A9和SA9-03、SA9-05或NC处理的细胞,加入到上室中。下室加入含有20%血清的培养基,放入37度培养箱中进行培养,诱导细胞穿下基质胶。培养36小时后,用棉签擦去基质胶和上室内的细胞,4%多聚甲醛固定20分钟,用0.5%结晶紫染料进行染色30分钟,用PBS清洗3遍,进行拍照,利用软件Image J进行细胞计数。进行迁移实验时不用提前铺基底膜基质胶,其他步骤与上述相同,其结果如图4N-4O所示。该结果表明,与NC对比,SA9-03和SA9-05均可以抑制S100A9介导的FLS的细胞侵袭和迁移,SA9-05的效果好于SA9-03。
(4)在低血清(1%)培养的FLS中加入1000nM SA9-03、SA9-05或NC序列和5μg/mLS100A9,37℃环境下孵育3天,实验过程中每天更换含有1000nM SA9-03、SA9-05或NC序列和5μg/mL S100A9的低血清(1%)培养基。使用基于膜联蛋白-V的磁珠分选检测凋亡细胞比例,结果表明:与NC对比,SA9-03和SA9-05均可以抑制S100A9介导的FLS凋亡抵抗,SA9-05的效果好于SA9-03,见图4P。
进一步地,本领域技术人员知悉,可以在不影响所述SA9-03和SA9-05序列的基本功能,即特异性结合和/或抑制S100A9的基础上,对其进行改造,修饰,从而获得对应的衍生物。例如所述SA9-03序列的衍生物包括:所述SA9-03序列的至少一个碱基被修饰获得的衍生物、与所述SA9-03序列的同源性在60%以上的序列、与所述SA9-03序列进行杂交的序列、由所述SA9-03序列进行转录的序列、所述SA9-03序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-03序列改造成的肽核酸;所述SA9-05序列的衍生物包括:所述SA9-05序列的至少一个碱基被修饰获得的衍生物、与所述SA9-05序列的同源性在60%以上的序列、与所述SA9-05序列进行杂交的序列、由所述SA9-05序列进行转录的序列、所述SA9-05序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-05序列改造成的肽核酸。
硫代磷酸酯修饰是最简单和广泛使用的可应用于增加核酸酶抗性的化学修饰,未经修饰的核酸适配体可以显示活性,但他们会被核酸酶迅速降解,因此作用有限。在寡核苷酸的磷酸酯骨架上,硫代磷酸酯键用硫原子取代了非桥键氧原子,使得核苷酸间键抵抗核酸酶的降解从而更稳定。肽核酸(peptide nucleic acids,PNA)是一类以多肽骨架取代糖磷酸主链的DNA类似物,以中性的肽链酰胺2氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余的与DNA相同。PNA可以通过Watson Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构,有很高的杂交稳定性、优良的特异序列识别能力、不被核酸酶和蛋白酶水解,并可以与配基相连共转染进入细胞。
因此,所述SA9-03序列的衍生物优选为所述SA9-03序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-03序列改造成的肽核酸;所述SA9-05序列的衍生物优选为所述SA9-05序列的骨架衍生出的硫代磷酸酯骨架序列、所述SA9-05序列改造成的肽核酸。本领域技术人员可以采用本领域的常规方法制备前述硫代磷酸酯骨架序列和肽核酸,在此不再累述。
优选地,所述至少一个碱基被修饰包括磷酸化、甲基化、氨基化、巯基化或同位素化,或被替换成锁核酸、非锁核酸或者乙二醇核酸、吗啉核酸、肽核酸、苏阿糖核酸或桥联核酸。优选地,可在所述SA9-03序列或其衍生物或所述SA9-05序列或其衍生物的3’端、5’端或中间碱基接功能基团。所述功能基团包括荧光基团、放射性基团、治疗性药物、生物素、地高辛、纳米发光材料、核酸物质或酶标记物中的至少一种。
本领域技术人员可以基于任何已知方法和技术,获得前述SA9-03序列的衍生物和SA9-05序列的衍生物。对SA9-03序列和SA9-05序列的衍生物进行上述相同的实验,其结果与SA9-03和SA9-05类似,其同样可以浓度依赖性抑制S100A9介导的FLS的趋化因子、炎症因子和抗原提呈相关基因的表达,降低S100A9介导的FLS的增殖、侵袭、迁移和凋亡抵抗。
基于实施例三中的实验可知,本发明中的S100A9适配体可以浓度依赖性抑制S100A9介导的FLS的趋化因子、炎症因子和抗原提呈相关基因的表达,降低S100A9介导的FLS的增殖、侵袭、迁移和凋亡抵抗。因此,本发明的S100A9适配体可以用于抑制S100A9介导的FLS的激活。
本领域技术人员进一步知悉,炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤中都有S100A9的高表达,且S100A9促进了这些疾病的发生和发展。S100A9主要与多种细胞表面的TLR4、RAGE、CD33、CD68、CD69或CD147结合,激活下游疾病相关信号通路。因此,针对S100A9的功能进行抑制是这些疾病的治疗策略。
为了进一步进行验证,发明人将SA9-03序列和SA9-05序列应用到多种响应S100A9刺激的细胞(如巨噬细胞、乳腺癌细胞、淋巴细胞和内皮细胞)进行实验,其结果证明SA9-03序列和SA9-05序列均可以浓度依赖的抑制S100A9介导的TLR4、RAGE、CD33、CD68、CD69或CD147下游通路的激活。S100A9与TLR4、RAGE、CD33、CD68、CD69或CD147结合后促进炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤的发生和发展。因此,本发明中的S100A9适配体可以用于S100A9相关的炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤的治疗。
例如,所述炎症性疾病可以包括自身炎症性疾病,例如系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、炎症性肠病、自身免疫性甲状腺病、皮肌炎、多发性硬化等等;以及非自身炎症性疾病,例如细菌、病毒、真菌、支原体、衣原体、立克次体等感染导致的炎症性的疾病。所述感染性疾病可以包括例如中枢神经系统、皮肤、肺、肝脏、肠道等疾病。所述心血管疾病括包括冠心病,例如动脉粥样硬化、心肌梗塞、血栓栓塞性疾病、风湿性心脏病等。所述神经系统疾病包括轻度认知障碍、弥漫性路易体病、突触核蛋白病、亨廷顿氏病、阿兹海默病和帕金森等。
所述肿瘤包括肺癌、脑瘤,消化器官肿瘤、子宫癌、睾丸癌、上颚癌、咽喉癌、舌癌、口腔癌、肉瘤、骨肉瘤、白血病、神经系统肿瘤、脑胶质瘤、胶质母细胞瘤、皮肤癌、皮肤附属器官癌和皮肤转移癌、淋巴瘤、髓母细胞瘤、母细胞瘤、脂肪肉瘤、神经内分泌肿瘤、滑膜细胞肉瘤、胃泌素瘤、类癌肿瘤、间皮瘤、胰岛细胞癌、神经鞘瘤、脑膜瘤、黑素瘤、听神经瘤、腺癌、淋巴样恶性肿瘤、上皮鳞状细胞癌、小细胞肺癌、非小细胞肺癌、鳞状细胞癌、腺癌肺癌、腹膜癌、肺鳞癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、甲状腺癌、膀胱癌、乳腺癌、结肠癌、直肠癌、前列腺癌、唾液腺癌、肾癌、外阴癌、肛门癌、阴茎癌、食管癌、胆道肿瘤和头颈部癌等。
实施例四、SA9-03和SA9-05在CIA小鼠模型中的治疗效果
使用牛II型胶原免疫6-8周龄雄性DBA/1小鼠,方法如下:在等体积完全弗氏佐剂中乳化牛II型胶原(2mg/mL),乳化剂中包含结核分支杆菌终浓度为2mg/kg。取100μL乳化剂在小鼠尾跟部进行单次皮下免疫。28天后,小鼠开始发病。分别给予PBS、SA9-05、NC序列、TNF单抗阿达木单抗(Adalimumab)、以及阿达木单抗与SA9-05或NC序列的组合。SA9-05或NC序列为尾静脉给药,剂量为30mg/kg,给药周期为一周两次。阿达木单抗为腹腔给药,剂量为5mg/kg,给药周期为一周两次。经过35天给药后,处死小鼠,评估上述给药对CIA小鼠的治疗效果。实验设计如图5A所示。治疗结束后对小鼠进行关节炎评分,结果如图5B所示;对踝关节切片进行苏木精-伊红染色后的组织学分析包括滑膜增生、骨破坏和软骨侵蚀的评分,结果如图5C-5E所示。这些结果一致表明,与NC序列对比,SA9-05可以显著抑制CIA小鼠模型的疾病进展、滑膜增生、骨破坏和软骨侵蚀。SA9-05的治疗效果与阿达木单抗类似,SA9-05与阿达木单抗联合给药可以更有效的抑制CIA小鼠的发病。
对SA9-03进行上述相同的实验,其结果与SA9-05类似,其同样可以显著抑制CIA小鼠模型的滑膜增生、骨破坏和软骨侵蚀。SA9-03的治疗效果与阿达木单抗类似,SA9-03与阿达木单抗联合给药可以更有效的抑制CIA小鼠的发病。
对SA9-03的衍生物和SA9-05的衍生物同样进行上述实验,其结果同样表明SA9-03的衍生物和SA9-05的衍生物同样可以显著抑制CIA小鼠模型的疾病进展、滑膜增生、骨破坏和软骨侵蚀,其治疗效果与阿达木单抗类似,并且其与阿达木单抗联合给药可以更有效的抑制CIA小鼠的发病。
在本发明的其他优选实施例中,采用其他类风湿性关节炎的治疗药物,比如传统疾病调节抗风湿药物如甲氨蝶呤、来氟米特、羟氯喹和柳氮磺吡啶、生物类疾病调节抗风湿药物如阿巴西普、阿达木单抗、阿那白滞素、培塞利珠单抗、依那西普、戈利木单抗、英夫利西单抗、利妥昔单抗、沙利鲁单抗和托珠单抗;靶向合成疾病调节抗风湿药物如巴瑞替尼、托法替布和乌帕替尼、非甾体类抗炎药如布洛芬、双氯芬酸、吲哚美辛、美洛昔康、塞来昔布等;免疫抑制剂,例如环丙酰胺、硫唑嘌呤、霉酚酸酯、环孢素等;糖皮质激素,如强的松、泼尼松、曲安奈德等,采用类似方法与SA9-03及其衍生物或SA9-05及其衍生物进行组合也能有效抑制CIA小鼠的发病。
在本发明的优选实施例中,通过SELEX筛选得到了特异性结合和/或抑制S100A9的适配体SA9-03和SA9-05(即SEQ ID NO.1所示序列和SEQ ID NO.2所示序列),筛选过程中选取His标签的S100A9为靶蛋白,固定在His标签蛋白纯化磁珠上,用于正向筛选。选取未固定S100A9的His标签蛋白纯化磁珠为对照,用于负向筛选。随着SELEX筛选的进行,富集文库与S100A9的结合有显著增多且不结合对照磁珠。筛选完成后测序得到的最优适配体SA9-03和SA9-05浓度依赖性结合S100A9且不与对照磁珠结合。在类风湿性关节炎中,S100A9作用于滑膜成纤维细胞(FLS),促进滑膜成纤维细胞的激活。SA9-03和SA9-05可以抑制S100A9介导的FLS的激活。胶原诱导的关节炎(collagen-induced arthritis,CIA)小鼠是常用的模拟人类风湿性关节炎动物模型。S100A9在CIA小鼠中水平升高且促进关节炎。SA9-03和SA9-05全身给药可以抑制CIA小鼠模型的疾病进展。SA9-03或SA9-05与TNF单抗阿达木单抗(Adalimumab)联合给药可以更有效的抑制CIA小鼠疾病进展。
基于以上公开的内容,本领域技术人员知悉,前述S100A9适配体(又可以叫做特异性结合和/或抑制S100A9的适配体)SA9-03和SA9-05以及其衍生物,可以用于S100A9的检测、纯化、体内成像、诊断和药物递送中的应用。前述S100A9适配体也可以抑制S100A9的功能,用于S100A9相关疾病的治疗。优选地,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤等。在本发明的一个优选实施例中,前述S100A9适配体在类风湿性关节炎中有很好的治疗效果。
进一步的,基于以上公开的内容,本领域技术人员进一步可以制备一种药物组合物,所述药物组合物包括所述S100A9适配体,所述药物组合物用于S100A9相关疾病的诊断或治疗。S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤等。进一步的,基于以上公开的内容,本领域技术人员进一步可以制备用于治疗类风湿性关节炎的药物组合物,所述药物组合物包括所述S100A9适配体和传统疾病调节抗风湿药物如甲氨蝶呤、来氟米特、羟氯喹和柳氮磺吡啶、生物类疾病调节抗风湿药物如阿巴西普、阿达木单抗、阿那白滞素、培塞利珠单抗、依那西普、戈利木单抗、英夫利西单抗、利妥昔单抗、沙利鲁单抗和托珠单抗;靶向合成疾病调节抗风湿药物如巴瑞替尼、托法替布和乌帕替尼、非甾体类抗炎药如布洛芬、双氯芬酸、吲哚美辛、美洛昔康、塞来昔布等;免疫抑制剂,例如环丙酰胺、硫唑嘌呤、霉酚酸酯、环孢素等;糖皮质激素,如强的松、泼尼松、曲安奈德等。所述S100A9适配体优选可以与阿达木单抗联合给药。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种S100A9适配体,其特征在于,包含以下序列中的至少一种:
(1)SEQ ID NO.1所示序列或其衍生物;
(2)SEQ ID NO.2所示序列或其衍生物;
SEQ ID NO.1:5’-GTTGCAACTGGGCTGACCGTGTTAAACCCG-3’;
SEQ ID NO.2:5’-ACCTGCACTTAGAGGGCATTAAACGCTGGG-3’;
所述S100A9适配体为特异性结合S100A9的适配体、抑制S100A9的适配体、或同时特异性结合和/或抑制S100A9的适配体。
2.根据权利要求1所述的S100A9适配体,其特征在于,所述SEQ ID NO.1所示序列的衍生物包括:所述SEQ ID NO.1所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ IDNO.1所示序列的同源性在60%以上的序列、与所述SEQ ID NO.1所示序列进行杂交的序列、由所述SEQ ID NO.1所示序列进行转录的序列、所述SEQ ID NO.1所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.1所示序列改造成的肽核酸;
所述SEQ ID NO.2所示序列的衍生物包括:所述SEQ ID NO.2所示序列的至少一个碱基被修饰获得的衍生物、与所述SEQ ID NO.2所示序列的同源性在60%以上的序列、与所述SEQ ID NO.2所示序列进行杂交的序列、由所述SEQ ID NO.2所示序列进行转录的序列、所述SEQ ID NO.2所示序列的骨架衍生出的硫代磷酸酯骨架序列、所述SEQ ID NO.2所示序列改造成的肽核酸。
3.根据权利要求2所述的S100A9适配体,其特征在于,所述至少一个碱基被修饰包括磷酸化、甲基化、氨基化、巯基化或同位素化,或被替换成锁核酸、非锁核酸或者乙二醇核酸、吗啉核酸、肽核酸、苏阿糖核酸或桥联核酸;和/或
在所述SEQ ID NO.1所示序列或其衍生物或所述SEQ ID NO.2所示序列或其衍生物的3’端、5’端或中间碱基接功能基团,所述功能基团包括荧光基团、放射性基团、治疗性药物、生物素、地高辛、纳米发光材料、核酸物质或酶标记物中的至少一种。
4.根据权利要求1-3中任意一项所述的S100A9适配体在制备检测S100A9的产品、纯化S100A9的产品、体内成像S100A9的产品、诊断S100A9相关疾病的产品、治疗S100A9相关疾病的产品、或递送药物至S100A9相关疾病部位的产品中的应用。
5.根据权利要求1-3中任意一项所述的S100A9适配体在制备S100A9相关疾病的药物中的应用,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。
6.一种试剂盒或检测芯片,其特征在于,包括根据权利要求1-3中任意一项所述的S100A9适配体,所述试剂盒或检测芯片用于检测S100A9、纯化S100A9、体内成像S100A9、诊断S100A9相关疾病、治疗S100A9相关疾病或递送药物至S100A9相关疾病部位。
7.一种药物组合物,其特征在于,包括根据权利要求1-3中任意一项所述的S100A9适配体;
所述药物组合物用于治疗S100A9相关疾病,所述S100A9相关疾病包括炎症性疾病、感染性疾病、自身免疫病、心血管疾病、神经系统疾病和肿瘤。
8.根据权利要求7所述的药物组合物,其特征在于,所述药物组合物包括用于治疗类风湿性关节炎的药物组合物,所述药物组合物进一步包括疾病调节抗风湿药物、生物类疾病调节抗风湿药物、靶向合成疾病调节抗风湿药物、非甾体类抗炎药、免疫抑制剂或糖皮质激素;
所述疾病调节抗风湿药物包括甲氨蝶呤、来氟米特、羟氯喹和柳氮磺吡啶;所述生物类疾病调节抗风湿药物包括阿巴西普、阿达木单抗、阿那白滞素、培塞利珠单抗、依那西普、戈利木单抗、英夫利西单抗、利妥昔单抗、沙利鲁单抗和托珠单抗;所述靶向合成疾病调节抗风湿药物包括巴瑞替尼、托法替布和乌帕替尼;所述非甾体类抗炎药包括布洛芬、双氯芬酸、吲哚美辛、美洛昔康、塞来昔布;所述免疫抑制剂包括环丙酰胺、硫唑嘌呤、霉酚酸酯、环孢素;所述糖皮质激素包括强的松、泼尼松、曲安奈德。
9.根据权利要求8所述的药物组合物,其特征在于,所述药物组合物包括所述S100A9适配体和阿达木单抗。
10.一种S100A9适配体的筛选方法,包括以下步骤:
S1、合成初始随机单链DNA文库、正向引物和反向引物,所述文库包括如SEQ ID NO.3所示的随机单链DNA序列,所述正向引物包括如SEQ ID NO.4所示序列,所述反向引物包括如SEQ ID NO.5所示序列;SEQ ID NO.3:5’-TGAGAATATGTAGACGATCC-(N)-CGGAGCTTCAAGATGATCTG-3’;SEQ ID NO.4:5’-TGAGAATATGTAGACGATCC-3’;SEQ ID NO.5:5’-CAGATCATCTTGAAGCTCCG-3’;其中N表示20~60个随机DNA寡核苷酸;
S2、选取S100A9为靶蛋白,固定在蛋白纯化磁珠上,用于正向筛选,并选取未固定S100A9的蛋白纯化磁珠为对照,用于负向筛选;
S3、将所述文库和所述S100A9进行孵育后洗涤,然后洗脱与S100A9结合的序列并取第一上清;
S4、对所述第一上清中富集的单链序列进行扩增和分离以获得富集单链文库;
S5、将步骤S4中获得的富集单链文库重复所述步骤S3和S4至少三轮之后,采用所述未固定S100A9的蛋白纯化磁珠作为对照与所述富集单链文库进行孵育后取第二上清;
S6、将所述步骤S5中的第二上清用于顺序执行步骤S3和S4之后,按照步骤S5,步骤S3和步骤S4的顺序重复执行所述步骤S5、步骤S3和步骤S4;直至选出与S100A9结合能力达到峰值且与所述对照磁珠无结合的富集文库并进行测序以获得所述S100A9适配体;
所述S100A9适配体包含以下序列中的至少一种:
(1)SEQ ID NO.1所示序列或其衍生物;
(2)SEQ ID NO.2所示序列或其衍生物;
SEQ ID NO.1:5’-GTTGCAACTGGGCTGACCGTGTTAAACCCG-3’;
SEQ ID NO.2:5’-ACCTGCACTTAGAGGGCATTAAACGCTGGG-3’;
所述S100A9适配体为特异性结合S100A9的适配体、抑制S100A9的适配体、或同时特异性结合和/或抑制S100A9的适配体。
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