CN116790607A - 结合igfbp-3蛋白的核酸适体及其应用 - Google Patents
结合igfbp-3蛋白的核酸适体及其应用 Download PDFInfo
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- CN116790607A CN116790607A CN202310058725.5A CN202310058725A CN116790607A CN 116790607 A CN116790607 A CN 116790607A CN 202310058725 A CN202310058725 A CN 202310058725A CN 116790607 A CN116790607 A CN 116790607A
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Abstract
本发明公开了结合IGFBP‑3蛋白的核酸适体及其应用,属于生物技术领域。本发明核酸适配体为活体SELEX筛选所得,序列如SEQ ID NO.1所示。本发明核酸适配体能够结合IGFBP‑3蛋白,具有高度特异性、化学性质稳定、易于制备保存、分子量小的特点,并且稳定性较体外筛选所得的是配体的体内稳定性有所提升。
Description
技术领域
本发明涉及生物技术领域,具体涉及结合IGFBP-3蛋白的核酸适体及其应用。
背景技术
核酸是生命的基础,特定核酸序列的翻译可以控制遗传特征、蛋白质表达以及细胞功能。此外,核酸不仅可以用作存储遗传信息,也可以对生物化合物进行特异性识别。当核酸分子在以单链形式存在时被称为适配体,它们可以折叠成特殊的三维结构,从而靶向某些生物分子。更具体地说,适配体有潜力成为一种非常有效的生物分析工具。此外,核酸适配体与抗体相比具有分子量小、稳定性好、易改造修饰、无免疫原性、制作周期短、可以通过人工合成等优势,免去了动物免疫、饲养、蛋白提取和纯化等一系列流程,因此核酸适配体是一种非常理想的分子探针。
IGFBP-3是出生后血液中含量最丰富的一种IGF结合蛋白。它与IGF结合,防止它们被降解,促进IGF在身体各部分的运输,对IGFs结构进行修饰从而改变IGFs与其特异性受体之间的相互作用。由于血清IGFBP-3具有生长激素(GH)依赖性,并与GH分泌量相关,因此其测定有助于GH异常分泌的检测。尤其是在儿童时期,IGFBP-3值在确定GH分泌和作用的变化(即原发性IGF缺乏状态)方面起着重要作用。IGFBP-3与IGFs的结合亲和力不受ALS的影响。“三元络合物”是IGFs在循环中的主要储存形式。所以IGFBPs可能通过减少游离IGF(在生物学上可与IGF受体相互作用)的数量来对抗IGF的作用。所以,IGFBPs具有抗增殖、抗有丝分裂和促凋亡作用。有研究表明,IGFBP-3不仅通过诱导细胞凋亡来触发生长抑制作用,而且在人乳腺癌、肾癌和肺癌细胞G1细胞周期阻滞中也有作用。IGFBP-3的这种作用,使得其在癌症病理中备受关注。因此,特异性结合IGFBP-3蛋白核酸适配体有助于对IGFBP-3的进一步研究。
通过指数富集系统进化配体(SELEX)和细胞-SELEX是目前核酸适配体筛选的主要方法,经过几轮筛选后,与靶标结合的序列会得到富集,随后将富集的序列进行测序、鉴定,得到特异性的核酸适配体;但由于分子间相互作用、微环境等的不同,体外筛选得到的核酸适配体与靶标结合构象通常与体内不同,这也使得核酸适配体无法在体内发挥很好的作用。因此,需要研究更好的筛选方法,例如活体SELEX,让核酸适配体在活体环境中进行筛选,所得到的核酸适配体拥有更好的体内稳定性。
发明内容
本发明的目的在于提供一种能够结合IGFBP-3蛋白的核酸适配体,该种核酸适配体具有高度特异性、化学性质稳定、易于制备保存、分子量小、亲和力高、稳定性强等特点。并且本发明核酸适配体是采用活体筛选所得,通过该方法得到的核酸适配体的稳定性较体外筛选所得的适配体的体内稳定性有所提升。
为达到上述发明目的,采用如下技术方案。
一种结合IGFBP-3蛋白的核酸适配体,核酸适配体序列包括如SEQ ID NO.1所示序列;
SEQ ID NO.1:
TGGATGTCAGTCCGGCACGCATTACCCCTGTTCCCCCTTGTTGTGACACATCC。
优选地,上述核酸适配体通过活体SELEX筛选得到。
优选地,上述核酸适配体的筛选文库为:
5’-GTTCGTGGTGTGCTGGATGT(N36)TGACACATCCAGCAGCACGA-3’(SEQ ID NO.2)。
优选地,上述核酸适配体的制备方法包括:
文库和引物的设计与合成;
核酸适配体筛选;
核酸适配体鉴定。
更优选地,核酸适配体筛选包括:
荷瘤小鼠模型构建;
向小鼠模型体内注入文库;
收集、扩增和纯化肿瘤组织细胞中核酸分子;
收集Cy5标记的核酸分子;
从文库中筛选高亲和力核酸适配体。
活体筛选所得的核酸适配体的稳定性较体外筛选所得的核酸适配体的体内稳定性有所提升。
本发明还公开了一种结合IGFBP-3蛋白的核酸适配体,包括上述核酸适配体SEQID NO.1的互补序列转录所得的RNA序列。
本发明还公开了一种结合IGFBP-3蛋白的核酸适配体,包括对上述核酸适配体进行修饰得到的序列。
优选地,修饰包括以下修饰方法的至少一种:
(1)磷酸化;
(2)甲基化;
(3)氨基化;
(4)巯基化;
(5)同位素化;
(6)用硫取代氧;
(7)用硒取代氧。
本发明还公开了一种结合IGFBP-3蛋白的核酸适配体,包括对上述核酸适配体进行改造得到的序列;改造包括以下改造方法的至少一种:
(1)在核酸适配体上连接荧光标记物;
(2)在核酸适配体上连接放射性物质;
(3)在核酸适配体上连接治疗性物质;
(4)在核酸适配体上连接生物素;
(5)在核酸适配体上连接地高辛;
(6)在核酸适配体上连接纳米发光材料;
(7)在核酸适配体上连接小肽;
(8)在核酸适配体上连接siRNA。
本发明还公开了一种结合IGFBP-3蛋白的核酸适配体的衍生物,包括以下至少一种:
(1)由上述核酸适配体骨架衍生出的硫代磷酸酯骨架序列;
(2)由上述核酸适配体设计合成的肽核酸序列。
本发明还公开了上述核酸适配体以及核酸适配体衍生物的用途,包括以下至少一种:
(1)检测IGFBP-3蛋白;
(2)定位成像表达IGFBP-3的细胞、组织或活体;
(3)捕获表达IGFBP-3的细胞或外泌体;
(4)偶联药物;
(5)靶向递送药物至肿瘤细胞;
(6)调控IGFBP3蛋白功能;
(7)制备IGFBP-3蛋白检测试剂;
(8)制备成像剂;
(9)制备药物载体;
(10)制备抗肿瘤药物。
本发明还公开了活体筛选在提高核酸适配体亲和力和/或稳定性中的用途。
与现有技术相比,本发明的有益效果为:
本发明核酸适配体通过活体SELEX筛选所得,具有高度特异性、化学性质稳定、易于制备保存、分子量小的特点;并且能够有效克服核酸适配体进入活体后,由于活体微环境、靶标构象的改变无法在体内稳定存在及与靶标结合发挥相应功能的问题。因此活体SELEX筛选所得核酸适配体的稳定性较体外筛选所得的是配体的体内稳定性有所提升,稳定性提升有助于进一步提高核酸适配体的亲和力,测试中本发明核酸适配体亲和力可达15nM。
附图说明
图1为SPR验证IGFBP3-L1与IGFBP3蛋白的亲和力的结果;
图2为流式细胞技术验证IGFBP3-L1与IGFBP3阳性细胞株结合情况的结果。
具体实施方式
这里将详细地对示例性实施例进行说明,以下示例性实施例中所描述的实施方式并不代表与本公开相一致的所有实施方式。相反,它们仅是与本公开的一些方面相一致的方法的例子。
下述实施例中的实验方法,如无特殊说明,均为常规方法,或按照制造厂商所建议的条件。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
特异性结合IGFBP3蛋白的核酸适配体的筛选
1、文库和引物的设计与合成
随机单链DNA文库:lib26;
SEQ ID NO.2:
5’-GTTCGTGGTGTGCTGGATGT(N36)TGACACATCCAGCAGCACGA-3’;
其中,“N36”表示36个任意的核苷酸碱基连接而成的序列。
用于筛选的引物信息如表1,由生工生物工程(上海)股份有限公司合成。
表1 引物及其序列
引物名称 | 序列(5’-3’) | 序号 |
lib26-S1 | GTTCGTGGTGTGCTGGATGT | SEQ ID NO.3 |
lib26-Cy5-S1 | Cy5-GTTCGTGGTGTGCTGGATGT | |
lib26-A2 | TCGTGCTGCTGGATGTGTCA | SEQ ID NO.4 |
lib26-A2-Biotin | Biotin-TCGTGCTGCTGGATGTGTCA |
其中,引物名称中的S代表正向引物,引物名称中A代表反向引物
文库以及引物由生工生物工程(上海)股份有限公司合成。合成后的引物分别用DPBS缓冲液(氯化钙0.1g/L,氯化钾0.2g/L,磷酸二氢钾0.2g/L,六水氯化镁0.1g/L,氯化钠8g/L,十二水磷酸氢二钠2.8915g/L;PH7.4,25℃)配制成100μM的贮存液,于-20℃保存备用。
2、核酸适配体筛选
1)荷瘤小鼠模型构建
将卵巢癌细胞ID8/OVCAR4细胞注射入C57小鼠/裸鼠腹腔构建腹腔转移原位模型;
2)活体筛选
取随机单链核苷酸文库,13000rpm离心10min,将文库离心到管底,用PBS缓冲溶液稀释至2μM,并分装到PCR管进行变复性处理。处理过程如下:PCR仪设定程序95℃持续10min,此步骤目的是使折叠的单链解开,然后4℃持续5min,然后在室温(25℃)下保持5min。
随后将处理后的文库(100μL)由小鼠尾静脉注射进入体内,随后对小鼠进行Cy5荧光监测,观察文库富集情况,总观察时间为1h,间隔为10min;并在监测结束后处死小鼠,收集肿瘤组织,将组织放到平皿中用PBS冲洗数次,用手术刀片交替划,将组织划碎成1-2mm的小块,将其移入2mL EP管中,加入1.5mL的无钙镁的PBS重悬组织,然后加入30μL胶原酶,混匀后室温孵5min左右,用移液器吹打组织直至变粘稠,将液体经200目的筛网去除大的组织,250g离心回收细胞,然后加入200μL红细胞裂解液充分混匀,4℃下孵育3min后离心去除上清,细胞沉淀用PBS清洗1-2遍。最后加入200μL细胞裂解液在4℃下与细胞孵育5min,离心后取出上清,标记为elution。
以elution中的核酸分子为模板,用PCR进行扩增。方法如下:将全部模板elution加入2mL PCR mix中混匀。将混合液分成100μL/管,加入到PCR管中,扩增条件如下:95℃预变性2min,95℃变性1min,60℃退火1min,72℃延伸1min,共34个循环,4℃保存。PCR mix的配方见表2。
表2 PCR mix配方
试剂 | 总体积1000μL |
超纯水 | 875μL |
dNTP | 20μL |
Pfu酶 | 4μL |
酶buffer | 100μL |
正向引物 | 0.5μL |
反向引物 | 0.5μL |
扩增产物用正丁醇纯化:收集所有的PCR产物于50mL尖底离心管中,加入约5倍体积的正丁醇,在旋涡混合器上震荡以充分混匀;台式离心机,9000rpm(转/min)于25℃离心10min;移弃上相(正丁醇),得到浓缩的PCR扩增产物,按体积比1:1加入TBE/尿素变性缓冲液,煮沸变性15 min使DNA变性,随后将所有的样品进行尿素变性聚丙烯酰胺凝胶电泳,400V电压下电泳至溴酚蓝到达胶底部,使加长的Cy5标记的链与反向的链分开,变性聚丙烯酰胺凝胶配方如表3所示。
表3变性聚丙烯酰胺凝胶配方
成分 | 用量 |
尿 素 | 3.78g |
40%聚丙烯酰胺 | 1.8mL |
5*TBE | 1.8mL |
ddH2O | 2.25mL |
10%APS | 60μL |
TEMED | 15μL |
切胶回收Cy5标记的链:将凝胶取出放在塑料膜上,Ex(nm):650,Em(nm):670检测带有Cy5标记的ssDNA;用干净的刀片将目的条带直接切下,将胶条转移至1.5mL EP管中并捣碎,加入1mL ddH2O后沸水浴10min将胶中的ssDNA转移至溶液中,离心去除胶的碎片,留上清。上清用正丁醇纯化,方法同“扩增产物纯化”步骤。得到DNA单链用3KD的透析袋透析过夜,即可作为下一轮筛选的文库。
筛选过程中用QPCR检测DNA单链文库富集程度的变化,当DNA单链文库的富集程度满足要求后,可进行靶蛋白垂钓和测定,在本实验中得到的靶蛋白为IGFBP3;然后用SPR检测DNA单链文库对IGFBP3蛋白识别能力的变化,当DNA单链文库对IGFBP3蛋白的识别能力满足要求,即筛选后的DNA单链文库与靶标的结合能力高于筛选起始投入的文库,将所得产物经克隆测序分析,最终得到核酸适配体。
所述筛选方法中,可逐轮增加筛选压力,以增进筛选核酸适配体的富集程度,缩短筛选进程。所述增加筛选压力包括减少投入的单链DNA文库的量和孵育时间。
3、核酸适配体鉴定
分析和鉴定多次筛选后得到的核酸适配体,将得到的富集文库产物经克隆测序分析后,挑选若干条序列由上海生工合成,检测亲和力。
在后续检测中,确定了1条序列具有很强的结合能力,将这条序列进行截短之后得到了核酸适配体SEQ ID NO.1:
5’-TGGATGTCAGTCCGGCACGCATTACCCCTGTTCCCCCTTGTTGTGACACATCC-3’,且验证后具有理想的结合IGFBP3蛋白的亲和力,将其命名为IGFBP3-L1。
试验例1
核酸适配体亲和力检测
委托苏州金唯智生物科技有限责任公司合成核酸适配体IGFBP3-L1,将其用DPBS缓冲液稀释成500nM。
1、将IGFBP3蛋白偶联到CM5芯片表面的第2通道,具体方法如下:先用50mM NaOH清洗芯片,进样20μL,流速10μL/分钟,然后用将等体积的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;0.4M水溶液)和NHS(N-羟基琥珀酰亚胺;0.1M水溶液)两个试剂混合后进样50μL活化芯片,流速5μL/分钟。将IGFBP3蛋白用pH3.6的10mM醋酸钠稀释至终浓度为50μg/mL后进样,进样体积50μL,流速5μL/分钟,IGFBP3蛋白偶联量为3000Ru。进样完成后,进乙醇胺封闭芯片,流速5μL/分钟,进样50μL。第1通道按照上述处理,偶联his小肽的步骤,活化和封闭的步骤完全一样,作为对照通道。
2、检测:使用表面等离子共振仪(GE Healthcare,型号:Biacore 8K)设定检测参数,稀释好的适配体样品流过通道,适配体的程序如下:进样30μL/分钟,时间3分钟,解离30μL/分钟,时间3分钟,再生1M NaCl 30μL/分钟,时间30秒,将稀释好的核酸适配体进样。
核酸适配体IGFBP3-L1与IGFBP3蛋白的亲和力检测数据见图1,KD值如表4所示,说明了对应的核酸适配体与靶标蛋白IGFBP3蛋白的结合能力。这些数据说明核酸适配体IGFBP3-L1用SPR仪均检测到与IGFBP3蛋白有很强的结合。
表4 核酸适配体与IGFBP3蛋白亲和力
核酸适配体 | 与IGFBP3蛋白的亲和力KD(nM) |
IGFBP3-L1 | 15nM |
3.核酸适配体与细胞的结合情况
用流式细胞技术对IGFBP3-L1与靶细胞的结合情况以及选择性进行表征,结果如图2所示。可见IGFBP3-L1与原代卵巢癌细胞株(PDC3)结合力优于对照正常卵巢上皮细胞(IOSE80)。
本发明的操作步骤中的常规操作为本领域技术人员所熟知,在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种结合IGFBP-3蛋白的核酸适配体,其特征在于,所述核酸适配体序列如SEQ IDNO.1所示;
SEQ ID NO.1:
TGGATGTCAGTCCGGCACGCATTACCCCTGTTCCCCCTTGTTGTGACACATCC。
2.如权利要求1所述的核酸适配体,其特征在于,所述核酸适配体还包括SEQ ID NO.1的互补序列转录所得的RNA序列。
3.一种结合IGFBP-3蛋白的核酸适配体,其特征在于,所述核酸适配体包括对权利要求1或2中所述的核酸适配体进行修饰得到的序列。
4.根据权利要求3所述的核酸适配体,其特征在于,所述修饰包括以下修饰方法的至少一种:
(1)磷酸化;
(2)甲基化;
(3)氨基化;
(4)巯基化;
(5)同位素化;
(6)用硫取代氧;
(7)用硒取代氧。
5.一种结合IGFBP-3蛋白的核酸适配体,其特征在于,所述核酸适配体包括对权利要求1或2中所述的核酸适配体进行改造得到的序列;所述改造包括以下改造方法的至少一种:
(1)在核酸适配体上连接荧光标记物;
(2)在核酸适配体上连接放射性物质;
(3)在核酸适配体上连接治疗性物质;
(4)在核酸适配体上连接生物素;
(5)在核酸适配体上连接地高辛;
(6)在核酸适配体上连接纳米发光材料;
(7)在核酸适配体上连接小肽;
(8)在核酸适配体上连接siRNA。
6.一种结合IGFBP-3蛋白的核酸适配体的衍生物,其特征在于,所述衍生物包括以下至少一种:
(1)由权利要求1或2中所述的核酸适配体骨架衍生出的硫代磷酸酯骨架序列;
(2)由权利要求1或2中所述的核酸适配体设计合成的肽核酸序列。
7.权利要求1-5任一项所述核酸适配体以及权利要求6所述核酸适配体衍生物的用途,包括以下至少一种:
(1)检测IGFBP-3蛋白;
(2)定位成像表达IGFBP-3的细胞、组织或活体;
(3)捕获表达IGFBP-3的细胞或外泌体;
(4)偶联药物;
(5)靶向递送药物至肿瘤细胞;
(6)调控IGFBP3蛋白功能;
(7)制备IGFBP-3蛋白检测试剂;
(8)制备成像剂;
(9)制备药物载体;
(10)制备抗肿瘤药物。
8.活体筛选在提高核酸适配体亲和力和/或稳定性中的用途。
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