CN112851811A - 一种cd44v6纳米抗体及其作为白血病研究试剂的应用 - Google Patents
一种cd44v6纳米抗体及其作为白血病研究试剂的应用 Download PDFInfo
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Abstract
本发明具体涉及一种CD44V6纳米抗体及其作为白血病研究试剂的应用。本发明研究提供了一种羊驼源的CD44V6单克隆抗体,包括靶点抗原羊驼免疫、纳米抗体快速筛选技术、纳米抗体大规模表达纯化。经验证,上述方案制备得到的CD44V6具有良好的诱导白血病细胞分化成熟的效果,可作为一种白血病研究试剂加以应用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种CD44V6的纳米抗体,所述纳米抗体的抗原蛋白及上述蛋白作为白血病研究试剂的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
近20年来重组抗体技术得到迅速的发展,抗体通过与其特定的抗原相结合,一方面用于建立检测抗原的酶联免疫试剂盒,另一方面可以直接结合靶抗原或者携带治疗药物实现在目的细胞上的特异性富集,或通过信号传导通路抑制其与天然配体的结合来影响细胞的生命活动。然而,尽管抗体药物应用范围如此之广,却也存在很多问题,比如研发周期长,生产成本高;难以大规模生产;易降解,储存成本高;容易被污染,维护成本费用高昂等等,使其在临床上的应用受到一定限制。
后来人们逐渐发现在羊驼外周血液中存在一种天然缺失轻链的抗体,该抗体只包含一个重链可变区(Variable domain of heavy chain of heavy chain antibody, VHH)和两个常规的CH2与CH3区。更重要的是单独克隆并表达出来的VHH 结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性,是目前已知的可结合目标抗原的最小单位。VHH晶体为2.5nm,长4nm,分子量只有15KDa,此也被称作纳米抗体(Nanobody,Nb)。VHH可溶性极高,不易聚集,能耐高温、强酸、强碱等致变性条件,适合于原核表达和各种真核表达系统,广泛用于开发治疗性抗体药物、诊断试剂、亲和纯化基质和科学研究等领域。纳米抗体具有独特的性质如分子量小、水溶性好、稳定性强、抗原识别能力强、易于生产等。因此,在体外诊断、肿瘤的诊断与治疗方面比其他抗体更具优势。如今,国外一些公司(如比利时的ablynx公司)和研究机构(如比利时的Muyldermans研究组) 针对一系列靶点(IL6R、TNF、IL17、CXCR4、HER2)和适应症(类风湿关节炎、银屑病、乳腺癌)对纳米抗体进行了深入的研究及广泛开发,一些产品分别进入临床前和临床开发阶段。
目前国内针对纳米抗体的研究较少,且均处于前期研究阶段尚无产品上市。急性骨髓性白血病(Acute myeloid leukemia,AML)是一种骨髓性干细胞异常增殖的血癌,由于干细胞受损,使得白血病细胞增殖、分化、成熟的能力下降,而停滞在细胞发育的不同阶段。未成熟细胞在体内无控制性增殖并积累,取代了正常骨髓,导致正常细胞数量减少,骨髓功能丧失。CD44为分子量80-90KD糖蛋白,含有20个外显子,其中由外显子6-15编码的V1-V10为可变体区,在一些特殊细胞种类中表达。CD44在急慢性白血病、恶性淋巴瘤、多发性骨髓瘤等许多血液系统恶性肿瘤均有表达,且急性髓系白血病(AML)各亚型均表达CD44V6 突变体分子。近年来有实验发现特异性抗CD44V6单克隆抗体或其配体透明质酸可逆转AML1至AML5型细胞的分化阻滞,触发白血病细胞的终末分化,提示CD44V6有可能成为AML诱导分化治疗的新靶点,其对应的抗体不仅用于细胞表面抗原和可溶性抗原的检测,也可以用于靶向抗原封闭或诱导白血病分化的启动靶点。
发明内容
针对上述研究背景,本发明目的在于提供一种CD44V6的纳米抗体,基于羊驼重链抗体的VHH单域抗体的特殊结构,兼具了传统抗体与小分子药物的优势,几乎完美克服了传统抗体的开发周期长,稳定性低,保存条件苛刻等缺陷,逐渐成为新一代治疗性生物医药与临床诊断试剂中的新兴力量。
为了实现上述技术目的,本发明提供以下技术方案:
本发明第一方面,提供一种CD44V6的纳米抗体,所述纳米抗体的抗原结合片段如SEQ ID NO:1所示,或其抗原结合片段包含SEQ ID NO:1所示序列。
针对CD44V6纳米抗体的开发,本发明首先针对抗原蛋白序列进行了筛选,采用上述抗原蛋白进行动物免疫能够获得稳定表达的纳米抗体。
另外,本发明还提供了基于基因工程的CD44V6抗原蛋白制备方法,通过构建重组菌株发酵培养,对发酵物进行分离纯化;其中,由于CD44V6含有二硫键和糖基化位点,因此选择可进行二硫键配对及具有糖基化修饰能力的毕赤酵母表达系统制备抗原。
本发明第二方面,提供编码所述CD44V6纳米抗体或SEQ ID NO:1所示抗原结合片段的核酸分子。
本发明第三方面,提供第一方面所述CD44V6纳米抗体的制备方法,所述制备方法包括动物免疫后获取抗体血清,通过反转录方式获取抗体序列并构建表达菌株。
上述动物免疫优选羊驼进行动物免疫,该纳米抗体没有传统的Fc段,CDR3 区比传统抗体更长,其CDR区的5个氨基酸变为亲水性氨基酸。通过重组抗原蛋白免疫羊驼,获取免疫羊驼后的抗体血清,设计兼并引物,反转录PCR,得到总 cDNA序列,转入到噬菌体展示文库中,通过亲和力和胰高血糖素拮抗性验证,得到候选抗体序列。最后通过筛选稳定表达的菌株实现所述纳米抗体的大量制备。
由于所述纳米抗体及抗原蛋白与CD44具有良好的亲和性,有望作为一种靶向诱导剂诱导药物集中于白血病细胞表面。基于该特性,本发明第四方面,还提供一种药物组合物,所述药物组合物包括第一方面所述CD44V6的纳米抗体和/ 或NO:1所示的多肽。
第五方面,提供一种药物,所述药物具有靶向基团修饰述靶向基团包括第五方面所述药物组合物。
第六方面,提供一种白血病模型研究试剂,所述研究试剂中包括第五方面所述药物组合物。
以上一个或多个技术方案的有益效果是:
本发明提供了一种CD44V6的纳米抗体,相比传统的单克隆抗体具有更小的分子量和稳定性,作为生物制剂应用于人体有望具有更好的生物利用度。所述纳米抗体具有良好的白血病细胞诱导分化作用,有望作为一种分化诱导剂参与白血病模型相关研究。另外,本发明还开发了所述CD44V6的纳米抗体的制备方法,可实现高纯度抗体的大量制备。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例3中所述纳米抗体扩增片段电泳图;
图2为所述CD44V6的测序结果;
图3为所述CD44V6中CDR3区的测序结果;
图4为实施例4中所述竞争法Elisa检测抗原抗体反应;
图5为实施例5中所述PAGE电泳检测抗体纯度与分子大小结果图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的,CD44V6能够触发白血病细胞的终末分化,是一种前景良好的诱导分化治疗剂。基于羊驼重链抗体的特殊结构有望获得尺寸更小、开发更为稳定的单克隆抗体。
本发明第一方面,提供一种CD44V6的纳米抗体,所述纳米抗体的抗原结合片段如SEQ ID NO:1所示,或其抗原结合片段包含SEQ ID NO:1所示序列。
优选的,所述纳米抗体还包括其修饰后的物质,所述修饰包括但不限于聚乙二醇(PEG)、抗生物素蛋白、链霉亲和素、各种分子,例如生物素、放射性同位素、荧光剂、酶、细胞毒性物质、抗肿瘤剂以及用它们修饰的第二抗体。其具体修饰方式可以采用目前公知的方式进行。
所述放射性同位素的例子包括18F,15O,13N,11C,82Rb,68Ga,198Au,199Au,32P,33P,125I,131I,123I,90Y,186Re,188Re,62Cu,64Cu,67Cu,47Sc,103Pb,109Pb,212Pb,71Ge,77As,105Rh,113Ag,119Sb,131Cs,143Pr,161Tb,177Lu,191Os,193Pt,197Hg等。放射性同位素可以通过螯合剂等直接或间接地通过已知方法连接或修饰。
所述螯合剂,例如,可以使用DTPA(二亚乙基三胺五乙酸)、DOTA(1,4,7,10- 四氮杂环十四烷-1,4,7,10-四乙酸)、DFO(去铁胺)等。
所述荧光剂的实例包括FITC(异硫氰酸荧光素)、罗丹明,藻红蛋白、藻蓝蛋白,别藻蓝蛋白,OPA(邻苯二甲醛)和氟胺。
所述酶的实例包括辣根过氧化物酶、β-半乳糖苷酶、萤光素酶和碱性磷酸酶。
所述细胞毒性物质的实例包括白喉A链、铜绿假单胞菌外毒素A,百日咳毒素、蓖麻毒蛋白A链、modesin毒素,α-sarcin,diandian,curcin,crotin,gelonin 或mitogellin。其实例包括克林霉素、苯霉素、线虫霉素、单端孢菌素、天花粉蛋白、细胞松弛素B、二羟基蒽indione、米托蒽醌、依米丁、秋水仙碱和皂草素。
所述抗肿瘤剂,并且其实例包括烷基化剂、抗代谢物、抗肿瘤抗生素、抗肿瘤植物成分、BRM(生物响应性调节剂)、血管生成抑制剂、细胞粘附抑制剂,基质金属蛋白酶抑制剂等。
优选的,本发明中,还提供一种通过基因工程制备CD44V6抗原蛋白的方法,通过构建重组菌株发酵培养,对发酵物进行分离纯化;其中,所述重组菌株为毕赤酵母。
进一步的,所述重组抗原蛋白的N端或C端具有标签。
本发明第二方面,提供编码单克隆抗体CD44V6或SEQ ID NO:1所示抗原结合片段的核酸分子。
优选的,所述核酸分子序列如SEQ ID NO:3所示。由于密码子简并性能够翻译并得到上述SEQ ID NO:1所示多肽序列的核酸分子也在本申请的保护范围内。
本发明第三方面,提供一种第一方面所述CD44V6纳米抗体的制备方法,所述制备方法包括动物免疫后获取抗体血清,通过反转录方式获取抗体序列并构建表达菌株。
优选的,所述免疫的动物包括猴子,兔子,狗,豚鼠,小鼠,大鼠,绵羊、山羊或羊驼,优选使用羊驼。
进一步的,所述抗原为SEQ ID NO:1所示的多肽修饰物,所述修饰包括采用载体蛋白进行偶联的方式,所述载体蛋白包括但不限于KLH,BSA或OVA。
进一步的,所述佐剂包括弗氏完全佐剂、弗氏不完全佐剂。
进一步的,所述产生抗体的器官包括但不限于脾脏或淋巴结。
本发明第五方面,提供一种药物组合物,所述药物组合物包括第一方面所述CD44V6的纳米抗体和/或NO:1所示的多肽。
优选的,所述药物组合物中,还包括药学上所必须的辅料。
本发明第六方面,提供一种药物,所述药物具有靶向基团修饰述靶向基团包括第五方面所述药物组合物。
本发明第七方面,提供一种白血病模型研究试剂,所述研究试剂中包括第五方面所述药物组合物。
优选的,所述白血病模型研究试剂用于诱导白血病细胞分化。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1 CD44抗原制备:
(1)蛋白信息:Uniprot Accession:https://www.uniprot.org/uniprot/P16070;物种名称:Homo sapiens(Human);蛋白长度:Q21-Q320;蛋白纯度: 90%;表达系统:大肠杆菌表达系统。
(2)实验内容:
1)CD44-pet28a重组载体获取:目的基因获取和构建由第三方公司合成,经测序结果比对,合成序列完全正确,符合实验设计。
2)CD44重组蛋白表达纯化:取50uL表达菌株接种至5mL的LB培养基中(含终浓度为50ug/mL卡那霉素),37℃,220rpm,过夜培养。将试管中5mL菌液接种至400mL的培养基中(共接种2瓶,每瓶400mL),加卡那霉素(母液浓度为50g/L)使其终浓度为50ug/mL,放置摇床,37℃,220rpm,培养3h。加IPT G至终浓度为0.5mM(IPTG母液浓度为500mM),37℃,220rpm,诱导表达4 h,用离心瓶收集菌液,14℃,6000rpm,离心1min,去上清,保留菌体,-20℃保存。
3)菌体处理:菌体重悬。向菌体中加10mL Buffer A重悬,将菌体从离心瓶转入离心管,再加10mL Buffer A将离心瓶涮洗一遍,转移至离心管中。破菌,加Buffer A定容至30mL,加入PMSF使其终浓度1mM,用超声破碎仪在冰浴的环境中破菌(300W,12min),12000rpm,4℃离心20min,收集上清,菌体沉淀待检。菌体沉淀用8M尿素溶解30min,12000rpm离心30min,去掉沉淀,保留上清,待纯化。
4)CD44重组蛋白纯化:利用亲和层析的方法纯化重组蛋白CD44,具体步骤如下:柱平衡。Buffer B平衡柱子,40mL/柱,平衡流速2.5mL/min;上样。上样流速0.8mL/min,用50mL烧杯收集流穿液;洗涤杂蛋白。Buffer C洗杂,300mL/ 柱,洗杂流速4mL/min;洗脱前准备。将管道中Buffer C液体排空,当柱面液体与介质界面平齐时,拔出柱塞。用Buffer D充满管道后,暂停蠕动泵,加大约 0.5mL的Buffer D于柱面上盖紧塞子。目的蛋白洗脱。第一步洗脱:Buffer D洗脱,6mL/柱,洗脱流速1.5mL/min,用10mL瓶子收集;第一步洗脱液量到一半时迅速换成Buffer E;第二步洗脱:Buffer E洗脱,8mL/柱,1.5mL/min,用20mL 瓶子收集;PAGE检测。
实施例2抗体制备
1、使用完全弗氏佐剂或者不完全弗氏佐剂乳化1-2mg共1.6mL抗原蛋白以供羊驼免疫。
2、羊驼选择:选择健康强壮、精神状态良好、体型适中的羊驼,挑选的羊驼毛色光亮,无受伤不适症状。挑选好动物,先预养1周左右以淘汰有些不合格的动物,使后期的实验能顺利进行。最终选择1-2岁健康母羊驼,编号7764。
3、免疫:挑选好羊驼并确保动物适合,记录耳号后开始免疫实验。每次免疫前抽取5mL血作为免疫效价检测使用。免疫时每次在羊驼颈部淋巴结附近分左右两侧注射,每侧注射0.8mL共1.6mL(约1-2mg)混合好的抗原。免疫后观察半小时确认羊驼状态良好,无不适症状。每2周免疫一次,一共进行4次免疫。
4、采血和细胞分离:在第4次免疫后间隔5-7天进行采血,采血从羊驼颈部静脉采取,取20-30mL血液,分3个采血管收集并分离淋巴细胞。
实施例3 CD44纳米抗体筛选
1、淋巴细胞分离:共取来淋巴细胞50mL,加入稀释液及分离液进行淋巴细胞分离,并用裂解液裂解残留的红细胞,之后加入Trizol(5mL)裂解淋巴细胞。
2、提RNA:根据RNA提取流程提取RNA,并用50uL RNase free-water溶解,取2uL测浓度,为790ng/uL,从A260/280来看达到RNA提取纯度,有少量氯仿残留,不过不影响转录,可以进行RNA转录。
3、转录cDNA:将RNA按照转录试剂盒步骤进行转录,转录10管,cDNA体积为400uL。
4、第一轮巢式PCR验证:取不同量的cDNA进行PCR验证以获得最终PCR模板体积。以此次cDNA为例,50μL PCR体系,建议使用3.5μL模板浓度,同时建议使用pfu酶。
5、第一轮巢式PCR大量扩增:按照50μL PCR体系,使用5μL模板浓度进行大量PCR,共PCR 1mL,通过2%的核酸胶跑胶结果来看,可以确定有两条带分别在1000bp和650bp左右,条带清晰,无杂带,可以进行第二轮pcr,割胶回收 650bp的条带作为模板进行第二轮巢式pcr。
6、第二轮巢式PCR大量扩增:以第一次pcr 650bp割胶回收产物为模板进行第二次PCR,通过第二次PCR可以获得400bp左右的条带,条带单一且清晰,可以进行下一步载体构建步骤,通过柱回收方法回收400bp纳米抗体片段。
实施例4 CD44纳米抗体建库:
1、通过本次对10管感受态进行电转,之后各取20μL混合均匀,取10μL梯度稀释涂布Amp平板用于库容测定,通过测定可以确定本次CD44纳米抗体库电转库容为2x109cfu/mL,可以进行库容插入率和正确率验证。
2、CD44纳米抗体库质检:随机挑选20个单菌落送测序,返回18个测序结果,另外2个没有信号,说明插入率为90%,正确率为100%,多样性为100%。
3、CD44抗原包被验证:本次CD44抗原使用大肠杆菌表达,带有his标签,因此可以将抗原包被并使用抗his的二抗进行检测。通过显色反应可以确定抗原可以包被在96孔板上,可以进行噬菌体淘选。
4、噬菌体淘选及phage Elisa:从第三轮固态淘选侵染平板各挑取94x2=188个单菌落,用于做phage Elisa,以M13作为阴性,652nm读数。
5、高通量测序验证:通过测序已返回的结果来看,测序结果均为纳米抗体,从返回的测序结果的多样性来看CDR3区存在8个差异性较大的抗体。
6、高通量筛选特异性VHH:首先使用抗原CD44(2μg/mL)进行包被实验,之后用表达的纳米抗体-myc进行孵育,目的是验证第一,纳米抗体为CD44特异性纳米抗体;第二,myc表达是否没有遮盖,可以进行正常显色。通过本次实验,显色很清楚,阴阳性区分明显,说明抗体特异性没有问题,可以直标HRP。
实施例5 CD44纳米抗体序列与抗体电泳图
1、纳米抗体序列:CD44V6纳米抗体序列:
(1)氨基酸序列(SEQ ID NO:1)
QVQLQESGGGLVQPGGSLRLSCTASGRIYEINTMAWYRQLPDKERELVAEVT WSTGAKRYSDSVKGRFTISSDNAKRTVRLQMRSLMPEDRGHYYCNARVVEA GIIQEGEFWGQGTQVTVSS
(2)DNA序列(SEQ ID NO:2)
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTC TCTCAGACTCTCCTGTACAGCCTCTGGAAGGATCTATGAAATCAATACAATG GCCTGGTACCGCCAGCTTCCAGATAAAGAGCGCGAACTGGTCGCAGAAGT GACTTGGAGTACTGGTGCGAAAAGGTATTCAGACTCCGTGAAGGGCCGAT TCACCATCTCCAGCGACAACGCCAAGAGAACGGTGCGTCTGCAGATGAGA AGTCTGATGCCTGAGGACAGGGGCCATTATTATTGTAATGCTCGTGTAGTTG AAGCCGGAATCATCCAGGAAGGGGAGTTTTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCT.
2、纳米抗体纯化与电泳鉴定
利用亲和层析的方法纯化重组蛋白CD44V6抗体,具体步骤如下:柱平衡。 BufferB平衡柱子,40mL/柱,平衡流速2.5mL/min;上样。上样流速0.8mL/min,用50mL烧杯收集流穿液;洗涤杂蛋白。Buffer C洗杂,300mL/柱,洗杂流速 4mL/min;洗脱前准备。将管道中Buffer C液体排空,当柱面液体与介质界面平齐时,拔出柱塞。用Buffer D充满管道后,暂停蠕动泵,加大约0.5mL的Buffer D于柱面上盖紧塞子。目的蛋白洗脱。第一步洗脱:BufferD洗脱,6mL/柱,洗脱流速1.5mL/min,用10mL瓶子收集;第一步洗脱液量到一半时迅速换成Buffer E;第二步洗脱:Buffer E洗脱,8mL/柱,1.5mL/min,用20mL瓶子收集;PAGE 电泳检测抗体纯度与分子大小。
实施例6 CD44V6纳米抗体诱导白血病细胞分化实验
为了了解CD44单克隆抗体对AML细胞有无诱导分化作用,对何种类型的白血病细胞有作用,且在多少浓度范围内效果最佳,本实施例设置实验如下:
1、方法:收集CD44V6纳米抗体(2μg/mL)作用0、72h的NB4细胞,800r/min 离心5分钟去上清,用预冷的PBS洗涤两次,并用PBS液悬起细胞沉淀,将细胞浓度调整为1×106/mL,将细胞悬液移至检测管中,每管加100μL PBS,并分别孵育PE标记的小鼠抗人CD11b及各同型对照抗体各20μL,4℃冰箱避光孵育 20min,孵育抗体后,PBS洗涤两遍,去除未结合的抗体,将200μL预冷的PBS 重新悬起细胞,用尼龙网过滤细胞后置于流式细胞仪,检测细胞表面抗原的表达。
2、结果与结论:
(一)细胞形态变化:NB4细胞培养后,细胞形态均向成熟方向改变。核浆比例减小,胞核变小,染色质颜色变深,核仁消失,肾型核及分叶核增多。提示CD44 抗体能诱导细胞形态向成熟方向分化。
(二)NBT反应:加纳米抗体后,细胞NBT阳性率升高,从原来的平均8.33%(6%、11﹪、8﹪)提高到平均的43.66%(39﹪、55﹪、37﹪)。提示CD44抗体诱导细胞功能成熟。
(三)细胞表型分析:加药后,细胞表面抗原CD11b有所升高,从对照组的平均9.0%(8%、12﹪、7﹪)上升到45%(42﹪、44﹪和49﹪)。提示CD44单克隆抗体能诱导白血病细胞表型向成熟分化。上述结果综合证明,本CD44单克隆抗体对AML细胞的形态、功能、细胞表型及原癌基因表达等方面均有诱导分化作用。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 姜国胜
<120> 一种CD44V6纳米抗体及其作为白血病研究试剂的应用
<130> 2010
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 122
<212> PRT
<213> CD44V6纳米抗体
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Ile Tyr Glu Ile Asn
20 25 30
Thr Met Ala Trp Tyr Arg Gln Leu Pro Asp Lys Glu Arg Glu Leu Val
35 40 45
Ala Glu Val Thr Trp Ser Thr Gly Ala Lys Arg Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Arg Thr Val Arg
65 70 75 80
Leu Gln Met Arg Ser Leu Met Pro Glu Asp Arg Gly His Tyr Tyr Cys
85 90 95
Asn Ala Arg Val Val Glu Ala Gly Ile Ile Gln Glu Gly Glu Phe Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 366
<212> DNA
<213> CD44V6纳米抗体
<400> 2
caggtgcagc tgcaggagtc tgggggaggc ttggtgcagc ctggggggtc tctcagactc 60
tcctgtacag cctctggaag gatctatgaa atcaatacaa tggcctggta ccgccagctt 120
ccagataaag agcgcgaact ggtcgcagaa gtgacttgga gtactggtgc gaaaaggtat 180
tcagactccg tgaagggccg attcaccatc tccagcgaca acgccaagag aacggtgcgt 240
ctgcagatga gaagtctgat gcctgaggac aggggccatt attattgtaa tgctcgtgta 300
gttgaagccg gaatcatcca ggaaggggag ttttggggcc aggggaccca ggtcaccgtc 360
tcctct 366
Claims (10)
1.CD44V6的纳米抗体,其特征在于,所述纳米抗体的抗原结合片段如SEQ ID NO:1所示,或其抗原结合片段包含SEQ ID NO:1所示序列。
2.如权利要求1所述CD44V6的纳米抗体,其特征在于,所述纳米抗体还包括其修饰后的物质,所述修饰包括但不限于聚乙二醇、抗生物素蛋白、链霉亲和素、各种分子,例如生物素、放射性同位素、荧光剂、酶、细胞毒性物质、抗肿瘤剂以及用它们修饰的第二抗体。
3.如权利要求2所述CD44V6的纳米抗体,其特征在于,所述放射性同位素的例子包括18F,15O,13N,11C,82Rb,68Ga,198Au,199Au,32P,33P,125I,131I,123I,90Y,186Re,188Re,62Cu,64Cu,67Cu,47Sc,103Pb,109Pb,212Pb,71Ge,77As,105Rh,113Ag,119Sb,131Cs,143Pr,161Tb,177Lu,191Os,193Pt,197Hg;
或,所述螯合剂,包括DTPA、DOTA、DFO;
或,所述荧光剂包括FITC、罗丹明,藻红蛋白、藻蓝蛋白,别藻蓝蛋白,OPA和氟胺;
或,所述酶的实例包括辣根过氧化物酶、β-半乳糖苷酶、萤光素酶和碱性磷酸酶;
或,所述细胞毒性物质的实例包括白喉A链、铜绿假单胞菌外毒素A,百日咳毒素、蓖麻毒蛋白A链、modesin毒素,α-sarcin,diandian,curcin,crotin,gelonin或mitogellin。其实例包括克林霉素、苯霉素、线虫霉素、单端孢菌素、天花粉蛋白、细胞松弛素B、二羟基蒽indione、米托蒽醌、依米丁、秋水仙碱和皂草素;
或,所述抗肿瘤剂,并且其实例包括烷基化剂、抗代谢物、抗肿瘤抗生素、抗肿瘤植物成分、BRM、血管生成抑制剂、细胞粘附抑制剂,基质金属蛋白酶抑制剂。
4.如权利要求1所述CD44V6的纳米抗体,其特征在于,一种通过基因工程制备CD44V6抗原蛋白的方法,通过构建重组菌株发酵培养,对发酵物进行分离纯化;其中,所述重组菌株为毕赤酵母;
进一步的,所述重组抗原蛋白的N端或C端具有标签。
5.编码单克隆抗体CD44V6或SEQ ID NO:1所示抗原结合片段的核酸分子;
优选的,所述核酸分子序列如SEQ ID NO:3所示,还包括由于密码子简并性能够翻译并得到上述SEQ ID NO:1所示多肽序列的核酸分子。
6.权利要求1-4任一项所述CD44V6单克隆抗体的制备方法,其特征在于,所述制备方法包括动物免疫后获取抗体血清,通过反转录方式获取抗体序列并构建表达菌株。
7.如权利要求6所述CD44V6单克隆抗体的制备方法,其特征在于,所述免疫的动物包括猴子,兔子,狗,豚鼠,小鼠,大鼠,绵羊、山羊或羊驼,优选使用羊驼;
优选的,所述抗原为SEQ ID NO:1所示的多肽修饰物,所述修饰包括采用载体蛋白进行偶联的方式,所述载体蛋白包括但不限于KLH,BSA或OVA;
优选的,所述佐剂包括弗氏完全佐剂、弗氏不完全佐剂;
优选的,所述产生抗体的器官包括但不限于脾脏或淋巴结。
8.一种药物组合物,其特征在于,所述药物组合物包括权利要求1-4任一项所述CD44V6的纳米抗体和/或NO:1所示的多肽;
优选的,所述药物组合物中,还包括药学上所必须的辅料。
9.一种药物,其特征在于,所述药物具有靶向基团修饰述靶向基团包括权利要求8所述药物组合物。
10.一种白血病模型研究试剂,其特征在于,所述研究试剂中包括权利要求9所述药物组合物;
优选的,所述白血病模型研究试剂用于诱导白血病细胞分化。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070292441A1 (en) * | 2004-06-10 | 2007-12-20 | Glover Nicholas R | Tumor Specific Antibody |
CN101152210A (zh) * | 2007-09-10 | 2008-04-02 | 山东省医学科学院基础医学研究所 | As2O3联合TSA在制备诱导白血病HL60细胞由部分分化向终末分化转变的药物中的应用 |
US20110182897A1 (en) * | 2008-06-05 | 2011-07-28 | Ablynx N.V. | Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases |
US20130137856A1 (en) * | 2010-07-16 | 2013-05-30 | Jan Steyaert | Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof |
WO2014025198A2 (ko) * | 2012-08-09 | 2014-02-13 | 주식회사 한독 | Lfa3 변이체 및 상기 변이체 또는 lfa3 cd2 결합영역과 이에 표적 특이적 폴리펩타이드가 연결된 융합단백질 및 그 용도 |
CN108473579A (zh) * | 2015-10-22 | 2018-08-31 | 埃博灵克斯股份有限公司 | Gitr激动剂 |
US20190293656A1 (en) * | 2016-09-19 | 2019-09-26 | University Of Southern California | Non-radioactive cytotoxicity assays |
-
2021
- 2021-01-28 CN CN202110118525.5A patent/CN112851811B/zh not_active Expired - Fee Related
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070292441A1 (en) * | 2004-06-10 | 2007-12-20 | Glover Nicholas R | Tumor Specific Antibody |
CN101152210A (zh) * | 2007-09-10 | 2008-04-02 | 山东省医学科学院基础医学研究所 | As2O3联合TSA在制备诱导白血病HL60细胞由部分分化向终末分化转变的药物中的应用 |
US20110182897A1 (en) * | 2008-06-05 | 2011-07-28 | Ablynx N.V. | Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases |
US20130137856A1 (en) * | 2010-07-16 | 2013-05-30 | Jan Steyaert | Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof |
WO2014025198A2 (ko) * | 2012-08-09 | 2014-02-13 | 주식회사 한독 | Lfa3 변이체 및 상기 변이체 또는 lfa3 cd2 결합영역과 이에 표적 특이적 폴리펩타이드가 연결된 융합단백질 및 그 용도 |
CN108473579A (zh) * | 2015-10-22 | 2018-08-31 | 埃博灵克斯股份有限公司 | Gitr激动剂 |
US20190293656A1 (en) * | 2016-09-19 | 2019-09-26 | University Of Southern California | Non-radioactive cytotoxicity assays |
Non-Patent Citations (2)
Title |
---|
JIANING LIU等: "Down-regulation of CD44 contributes to the differentiation of HL-60 cells induced by ATRA or HMBA", 《CELL MOL IMMUNOL.》 * |
韩玉春: "抗CD44纳米抗体的制备与鉴定", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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