CN112898425B - 一种amh纳米抗体、试剂盒及在生殖领域的应用 - Google Patents
一种amh纳米抗体、试剂盒及在生殖领域的应用 Download PDFInfo
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Abstract
本发明具体涉及一种AMH纳米抗体、试剂盒及在生殖领域的应用。AMH与人体内生殖相关的激素具有密切的联系,通过检测血清中AMH的含量可用于多种疾病的诊断或生殖功能相关的评价。目前基于抗原抗体原理进行检测的产品中,所述抗体大多鼠源型抗体,临床检测效果并不稳定。基于上述现状,本发明提供了一种AMH纳米抗体,所述纳米抗体具有良好的检测特异性和亲和力,可应用多种生殖相关疾病的检测。
Description
技术领域
本发明属于纳米抗体开发技术领域,具体涉及一种AMH的纳米抗体、所述纳米抗体的编码基因、所述纳米抗体的抗原蛋白以及上述物质在药物组合物、药物及检测试剂盒中的应用。
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。
女性生育能力与其体内各类生殖激素和卵巢储备功能有着密切的联系。卵巢储备功能是卵巢产生卵子数量和质量的潜能,间接反映卵巢的功能,卵巢储备功能与生育和不孕症诊治关系密切,高效、准确的检测为临床医生全面了解女性的卵巢储备功能提供重要的参考信息,这对于科学制定个体化的生育计划,提高女性生育率具有重要意义。AMH对卵巢储备功能及卵泡发育发挥着重要的作用,能够反映整个生命周期的卵泡活性。女性AMH水平随着卵巢储备功能的变化而变化,AMH水平越高,说明卵泡数量越多,卵巢储备功能强,女性生育能力就较强。而随着年龄及各种因素逐渐消耗,AMH浓度也会随之降低,就代表着卵巢储备功能降低,也预示着女性生育力的减弱,直到绝经后无法测出。因此,检测AMH水平可以预测女性生育能力。
临床上,血清AMH水平的检测除了可用于卵巢功能储备的评估,还可用于控制性超排卵(COH)效果的预测、卵巢过度刺激(OHSS)的预测、多囊卵巢综合征(PCOS)的诊断和预后、卵巢颗粒细胞瘤的诊断和管理、男性性早熟和青春期延迟的诊断和鉴别诊断、男童的隐睾症和无睾症的诊断和儿童双性状态的鉴别诊断。AMH的检测具有广阔的市场前景,其诊断试剂盒市场开发潜力巨大。目前人AMH检测试剂盒国内以深圳市亚辉龙生物科技有限公司和广州市康润生物科技有限公司生产的抗缪勒氏管激素(AMH)定量检测试剂盒(酶联免疫法)为主,进口以贝克曼的AMH Gen II Assay和罗氏的电化学发光检测Elecsys AMH为主。针对上述现状,发明人认为,上述检测抗体均为传统的鼠源抗体,存在半衰期段、人源化之后抗原抗体亲和力减弱的缺陷,作为一种检测试剂的假阳性概率较高,无法满足临床检测血清中AMH的要求。
发明内容
针对上述研究背景,本发明目的在于提供一种能够满足临床检测的AMH相关抗体,基于该目的,本发明联想到提供一种AMH的纳米抗体,所述纳米抗体优选为AMH的骆驼源抗体。所述骆驼源单独克隆表达出来的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。
基于上述技术目的,本发明提供以下技术方案:
本发明第一方面,提供一种AMH的纳米抗体,所述纳米抗体的序列如SEQ ID NO:1所示。
本发明提供的上述纳米抗体相比传统单克隆抗体具有更小的尺寸,并且具有良好的结合特异性,作为检测抗体相比现有的鼠源抗体更加稳定,亲和力得到显著的提升。
发明第二方面,提供编码第一方面所述纳米抗体的基因序列,或由于密码子简并性能够翻译的到第一方面所述纳米抗体氨基酸序列的基因序列。基于该基因序列,本领域技术人员可通过常规的基因工程手段获得第一方面所述纳米抗体,另外,本发明还提供一种基于动物免疫手段获取上述纳米抗体的方法。
因此,本发明第三方面,提供第一方面所述AMH纳米抗体的抗原蛋白,所述纳米抗体的抗原结合片段如SEQ ID NO:3所示,或其抗原结合片段包括SEQ ID NO:3所示序列。
本发明第四方面,提供第一方面所述AMH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。
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AMH的纳米抗体和/或SEQ ID NO:3所示的多肽。
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,提供一种检测试剂盒,所述检测试剂盒中包括第一方面所述AMH纳米抗体或第三方面所述AMH纳米抗体的抗原结合蛋白。
本发明第八方面,提供第七方面所述检测试剂盒在生殖领域的应用。
以上一个或多个技术方案的有益效果是:
1、本发明提供的AMH纳米蛋白具有分子量小、特异性良好的特点,基于该特性,所述纳米蛋白作为检测抗体能够有效降低假阳性的概率,作为药物靶向基团不会显著增加药物的分子量,影响药物的生物利用度,其制备工艺简单,适用于临床检索。
2、本发明提供的AMH抗原蛋白的制备方法中,根据AMH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蛋白,通过添加标签减少了后续纯化步骤,能够分离得到纯度大于95%的重组抗原蛋白,并且结构稳定,特异性良好。
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PCR鉴定AMH纳米抗体表达质粒电泳图;
图2为实施例4中AMH纳米抗体表达质粒的测序结果图;
图3为实施例5中纯化包被纳米抗体电泳结果图;
图4为实施例6中AMH的ELISA方法检测的标准曲线。
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,血清AMH水平的检测对于多种疾病的诊断具有重要的意义,目前基于抗原抗体结合效应进行检测的抗体多为鼠源抗体,存在临床应用不稳定的缺陷。为了解决如上的技术问题,本发明提出了一种AMH的纳米抗体。
本发明第一方面,提供一种AMH的纳米抗体,所述纳米抗体的序列如SEQ ID NO:1所示。
优选的,所述纳米抗体中,没有Fc段,CDR3区比传统抗体更长,其CDR区的5个氨基酸为亲水性氨基酸。
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。
本发明第二方面,提供编码第一方面所述纳米抗体的基因序列,或由于密码子简并性能够翻译的到第一方面所述纳米抗体氨基酸序列的基因序列。
优选的,所述基因序列如SEQ ID NO:2所示。
本发明第三方面,提供第一方面所述AMH纳米抗体的抗原蛋白,所述纳米抗体的抗原结合片段如SEQ ID NO:3所示,或其抗原结合片段包括SEQ ID NO:3所示序列。
优选的,本发明中,还提供一种通过基因工程制备AMH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。
本发明第四方面,提供第一方面所述AMH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:3所示的多肽修饰物。
进一步的,所述抗原为抗原蛋白的佐剂,包括弗氏完全佐剂、弗氏不完全佐剂。
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AMH的纳米抗体和/或SEQ ID NO:3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,提供一种检测试剂盒,所述检测试剂盒中包括第一方面所述AMH纳米抗体或第三方面所述AMH纳米抗体的抗原结合蛋白。
优选的,所述检测试剂盒为Elisa试剂盒,所述Elisa试剂盒中,还包括显色剂、终止试剂、缓冲液中的一种或几种。
本发明第八方面,提供第七方面所述检测试剂盒在生殖领域的应用;
优选的,所述生殖领域的应用包括但不限于卵巢储备功能评价、控制性超排卵(COH)效果的预测、卵巢过度刺激(OHSS)的预测、多囊卵巢综合征(PCOS)的诊断和预后、卵巢颗粒细胞瘤的诊断和管理、男性性早熟和青春期延迟的诊断和鉴别诊断、男童的隐睾症和无睾症的诊断和儿童双性状态的鉴别诊断。
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1AMH重组蛋白表达质粒
(1)蛋白信息:Uniprot Accession:https://www.uniprot.org/uniprot/P0397
1;物种名称:Homo sapiens(Human);蛋白长度:R26-R560;蛋白纯度:90%;蛋白总量:5mg;表达系统:大肠杆菌表达系统。
(2)实验内容:AMH-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。AMH重组蛋白表达纯化:1)AMH重组蛋白诱导表达: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IPTG至终浓度为0.2mM(IPTG母液浓度为500mM),16℃,220rpm,诱导表达16h,用离心瓶收集菌液,14℃,6000rpm,离心1min,去上清,保留菌体,-20℃保存。
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,待纯化,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。
AMH重组蛋白纯化:利用亲和层析的方法纯化重组蛋白AMH,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;分别取超声破碎离心后的菌体,2中收集的流穿液、5中第一步洗脱和第二步洗脱样送电泳PAGE检测。
实施例2免疫羊驼
1、抗原准备:每次免疫前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、抗原免疫: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岁母羊驼。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。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。ELISA检测效价结果。结果表明连续三次免疫后,血清效价持续升高,说明免疫反应较好发生,并维持在较高的水平;第三次免疫后,血清效价达到107。免疫反应良好,血清以及外周血单核细胞(PBMC)能够满足后续实验需求。
实施例3AMH纳米抗体制备与筛选鉴定
AMH纳米抗体筛选: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。提RNA:根据RNA提取流程提取RNA,并用100μL RNase free-water溶解,取2μL测浓度,为244.5ng/μL,从A260/280来看达到RNA提取纯度,可以进行RNA转录。将RNA按照转录试剂盒步骤进行转录,转录10管,cDNA体积为400μL。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5μL模板浓度,同时建议使用pfu酶。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(图1),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。AMH抗体库质检:从电转平板上挑选阳性单菌落,用于菌落PCR验证,通过菌落PCR可以看出在400bp均有非常清晰的条带,说明插入率为100%,可以进行下一步正确率的验证。将这20株单菌落,测序验证正确率,正确率100%。说明本次建库库容108cfu/mL,插入率100%,正确率100%。可以进行下一步筛库。
实施例4AMH纳米抗体特异性检测
1、AMH抗原包被验证:AMH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。
2、高通量测序验证:通过测序结果来看,测序结果均为纳米抗体(图2),从返回的测序结果的多样性来看CDR3区存在12个差异性较大的抗体,进行进化树分析,通过分析纳米抗体不同类后,下一步对目标纳米抗体表达实验并测亲和力,如果亲和力不够可以做体外成熟用于提高亲和力。
3、高通量筛选特异性VHH:首先使用抗原AMH(2μg/mL)进行,包被实验,之后用表达的纳米抗体-myc标签进行孵育,目的是验证第一,纳米抗体为AMH特异性纳米抗体,第二,myc标签表达是否遮盖,是否可以进行正常显色。通过本次实验,显色很清楚,阴阳性区分明显,其中有9个纳米抗体是特异性的纳米抗体。之后进行标记HRP,进行亲和力和活性验证。
实施例5纳米抗体的诱导表达提取、纯化与鉴定
1、诱导表达:挑取上述单克隆菌落于LB-A培养基中,37℃振荡培养过夜;次日,取该菌液按照1:100比例加入100mL新鲜LB-A培养基,37℃振荡培养3h;至菌液OD600=0.8左右,加入终浓度为1mM IPTG,30℃诱导过夜;第三日,8000rpm,离心10min收集菌体,加入1.5mL的预冷TES缓冲液重悬沉淀;冰浴2min后,轻柔振荡30s,重复此循环6次;加3.0mLTES/4(将TES用水稀释4倍),轻柔振荡30s后,冰浴静置2min,同样重复振荡和静置步骤共6次;9000rpm,4℃离心10min,收集约4.5mL的上清(周质提取物),将上清进行蛋白电泳分析。
2、纯化和鉴定:将IMAC Sepharose重悬后,取2mL加入到重力柱内,静置30min,使sepharose自然沉降于重力柱底部,流出保存缓冲液。加入2倍柱体积的硫酸镍溶液,按照约8s/滴的流速流出硫酸镍溶液;加入10倍柱体积的平衡缓冲液平衡并洗涤sepharose,流速维持不变;将样品使用平衡缓冲液2倍稀释后,加入重力柱中,调节流速为6s/滴,收集穿透液;加入10倍柱体积洗涤缓冲液洗涤sepharose,维持流速不变,收集洗涤液;加入3倍柱体积的洗脱缓冲液,流速维持在6s/滴,收集含有目的蛋白的洗脱液;最后依次加入10倍柱体积的平衡缓冲液、10倍柱体积的纯水和10倍柱体积的20%乙醇洗涤sepharose,并最终保留4mL的20%乙醇来保存柱子。上述收集的样品分别进行SDS-PAGE检测(图3)。
3、关键步骤1:装好的镍柱可以置4℃保存数月,使用洗脱缓冲液洗脱目的蛋白后,尽可能使用500mM咪唑洗涤重力柱,确保无杂蛋白残留。关键步骤2:镍柱在使用超过10次以上,请务必执行镍柱重生:①使用5倍柱体积的0.5M EDTA溶液洗涤重力柱,至sepharose发白;②使用10倍柱体积的纯水溶液洗涤重力柱;③使用10倍柱体积的0.1M NaOH洗涤重力柱;④使用10倍柱体积的纯水溶液洗涤重力柱;⑤使用10倍柱体积的平衡缓冲液洗涤重力柱,可在此步骤前加50mM咪唑洗涤重力柱,来降低杂蛋白的非特异性的低吸附。
4、纳米抗体序列
(1)氨基酸序列(SEQ ID NO:1所示)
QVQLEVESGGGLVQAGGSLRLSCAASGRILSNYGMGWFRQAPGKEREFVAAISRSGGSTYYADSVKGRFTISRDNAKNTAYLQMNSLEPEDTAIYYCATRPMLGVIDNKNDYDYWGQGTQVTVSS
(2)DNA序列(SEQ ID NO:2所示)
CAGGTGCAGCTGGAGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCATCTTGAGTAATTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTGGCAGCTATTAGTCGGAGTGGTGGGAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGTCTGGAACCTGAGGACACGGCCATTTATTACTGTGCAACTCGTCCTATGTTAGGGGTAATTGATAATAAAAATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCT
实施例6可溶性AMH的ELISA方法建立
取出两条板条,固定在板子上。按照低浓度到高浓7个浓度,纵向加液体,每孔加100μL,放入自封袋,放入37℃培养箱静置1h。开始洗板,洗五次。洗好的板条,每孔加入100μL酶标二抗(1:2500),将板条放在自封袋,37℃培养箱静置1h。开始洗板,洗五次。每孔加入TMB显色液100μL,室温显色6分钟,加终止液100μL。使用酶标板设置波长450,进行读数,四参数Logistic曲线拟合,描述标准曲线(图4)。计算公式:y=(A-D)/[1+(x/C)^B]+D。
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AMH纳米抗体、试剂盒及在生殖领域的应用
<130> 2010
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 125
<212> PRT
<213> AMH纳米抗体
<400> 1
Gln Val Gln Leu Glu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Leu Ser Asn
20 25 30
Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Ser Arg Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Ala Thr Arg Pro Met Leu Gly Val Ile Asp Asn Lys Asn Asp Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 375
<212> DNA
<213> AMH纳米抗体
<400> 2
caggtgcagc tggaggtgga gtctggggga ggcttggtgc aggctggggg ctctctgaga 60
ctctcctgtg cagcctctgg acgcatcttg agtaattatg gcatgggctg gttccgccag 120
gctccaggga aggagcgtga atttgtggca gctattagtc ggagtggtgg gagcacatac 180
tatgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gaacacggcg 240
tatctgcaaa tgaacagtct ggaacctgag gacacggcca tttattactg tgcaactcgt 300
cctatgttag gggtaattga taataaaaat gactatgact actggggcca ggggacccag 360
gtcaccgtct cctct 375
Claims (12)
1.一种抗AMH的纳米抗体,其特征在于,所述纳米抗体的序列如SEQ ID NO:1所示。
2.如权利要求1所述抗AMH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,所述修饰包括聚乙二醇、抗生物素蛋白、链霉亲和素、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所述抗AMH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;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。
4.如权利要求3所述抗AMH的纳米抗体,其特征在于,所述螯合剂,为DTPA、DOTA或DFO。
5.如权利要求2所述抗AMH的纳米抗体,其特征在于,所述荧光剂为FITC、罗丹明,藻红蛋白、藻蓝蛋白,别藻蓝蛋白,OPA或氟胺。
6.如权利要求2所述抗AMH的纳米抗体,其特征在于,所述酶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。
7.如权利要求2所述抗AMH的纳米抗体,其特征在于,所述细胞毒性物质包括白喉A链、铜绿假单胞菌外毒素A,百日咳毒素、蓖麻毒蛋白A链、curcin,crotin,gelonin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、米托蒽醌、依米丁、秋水仙碱和皂草素。
8.如权利要求2所述抗AMH的纳米抗体,其特征在于,所述抗肿瘤剂包括烷基化剂、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。
9.一种药物组合物,其特征在于,所述药物组合物包括权利要求1-8任一项所述抗AMH的纳米抗体。
10.如权利要求9所述药物组合物,其特征在于,所述药物组合物中还包括药学上所必须的辅料。
11.一种检测试剂盒,其特征在于,所述检测试剂盒中包括权利要求1-8任一项所述抗AMH纳米抗体。
12.如权利要求11所述的检测试剂盒,其特征在于,所述检测试剂盒为Elisa试剂盒,所述Elisa试剂盒中,还包括显色剂、终止试剂、缓冲液中的一种或几种。
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