CN114702573A - 乙肝病毒表面s蛋白高亲和力纳米抗体及其应用 - Google Patents
乙肝病毒表面s蛋白高亲和力纳米抗体及其应用 Download PDFInfo
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Abstract
本发明提供乙肝病毒表面S蛋白高亲和力纳米抗体及其应用。本发明涉及与乙肝病毒表面小S蛋白特异性结合的骆驼源纳米抗体及其抗原结合片段,具体涉及能以高亲和力结合HBV ADR亚型表面小S蛋白的骆驼源纳米抗体或其抗原结合片段,其能够用于检测、诊断由HBV引起的感染,对治疗由HBV感染引起的肝炎肝癌及其并发症具有重要意义。
Description
技术领域
本发明属于生物技术、免疫检测和生物医药领域,具体地说,涉及乙肝病毒表面S蛋白高亲和力纳米抗体及其应用。
背景技术
乙型肝炎病毒(Hepatitis B Virus,HBV)是一种嗜肝病毒属DNA病毒。具有强烈的嗜肝性、高变异性、致癌和难以清除等特点。HBV主要通过血液、体液和母婴途径传播,潜伏期一般为30~180天。急性感染症状包括呕吐、黄疸病、疲劳、小便黄赤和急性腹痛等,此外,机体免疫反应低下或病毒变异导致的免疫逃逸则会使其转为慢性感染,逐步发展为肝硬化和肝癌等并发症。
HBV的表现型是其血清亚型(Subtype),由外膜主蛋白上的一些残基决定,包含众多基因型(HBV-A、HBV-C和HBV-D),具体包括ADW,ADY,AYW,ADR亚型。ADR亚型主要分布在东亚地区,与这一地区流行的C基因型一致。而ADW亚型在我国较为少见,但在世界范围内广泛分布,与基因型D型一致。
目前控制HBV传播的主要手段是疫苗接种,而对于慢性乙肝患者的治疗则主要通过使用干扰素和小分子药物(拉米夫定,阿德福韦酯,替比夫定,恩替卡韦,替诺福韦酯等)进行抗病毒治疗,然而上述的药物仅能遏制HBV的复制但无法彻底清除病毒感染。纳米抗体(Nanobody)是只含有重链抗体抗原结合域VHH的单域抗体,与传统多克隆抗体、单克隆抗体和单链抗体相比具有诸多明显优势,比如体积小,可以穿过常规抗体无法进入的组织和器官(如鞘膜、脊髓、大脑等)中;稳定性强,无需冷链运输和冷藏保存;免疫原性低,易于进行人源化改造。
发明内容
本发明的目的是提供乙肝病毒表面S蛋白高亲和力纳米抗体及其应用。
本发明构思如下:HBV外膜上的可识别抗原为S蛋白,一般分为大、中、小三种表面S蛋白。本发明将HBV表面小S蛋白作为靶标,通过构建噬菌体展示纳米抗体免疫文库、生物淘选研制出可识别HBV的骆驼源高亲和力纳米抗体,为乙型肝炎机理的进一步研究奠定基础,也为乙肝临床检测提供潜在的新选择。
为了实现本发明目的,第一方面,本发明提供一种乙肝病毒表面S蛋白高亲和力纳米抗体或其抗原结合片段,其高变区CDR1、CDR2和CDR3分别选自SEQ ID NO:9-15、SEQ IDNO:16-22和SEQ ID NO:23-30任一所示的氨基酸序列。
所述抗原结合片段为Fv、Fab、Fab'、scFv、F(ab')2、多价化或多特异片段。
进一步地,本发明的乙肝病毒表面S蛋白高亲和力纳米抗体或其抗原结合片段,其氨基酸序列如SEQ ID NO:1-8任一所示;或者
SEQ ID NO:1-8任一所示序列经截短得到的氨基酸序列;或者,
将SEQ ID NO:1-8任一所示序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的抗体或抗原结合片段。
本发明的纳米抗体与HBV具有纳摩尔级别的亲和力。
第二方面,本发明提供一种基因工程抗体,其包含所述的抗体或其抗原结合片段。
其中,所述基因工程抗体包括但不限于人源化抗体、嵌合抗体、多价化或多特异性抗体。
第三方面,本发明提供一种融合蛋白,其包含所述的抗体或其抗原结合片段或所述的基因工程抗体。
进一步地,所述融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。
第四方面,本发明提供一种偶联物,其包含所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白。
进一步地,所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP,或其组合。
第五方面,本发明提供一种核酸分子,其编码所述的抗体或其抗原结合片段,或编码所述的基因工程抗体,或编码所述的融合蛋白,或编码所述的偶联物。
其中,所述核酸分子可以是RNA、DNA或cDNA。
第六方面,本发明提供含有所述核酸分子的生物材料,所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第七方面,本发明提供一种药物组合物,其活性成分为所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物。
所述药物组合物可以是外用型、皮下注射型、血管输入型,或其组合。
优选地,所述药物组合物还包括药用赋形剂或载体。
第八方面,本发明提供所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物在制备用于预防、治疗和/或诊断HBV感染、相关科学研究的生物制品中的应用。
第九方面,本发明提供所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物的以下任一应用:
1)用于制备预防或治疗由HBV感染以及由其感染所致相关疾病的药物;
2)用于制备HBV检测试剂或试剂盒;
3)检测HBV抗原;
4)阻断HBV感染;
5)消杀HBV颗粒;
6)诊断HBV引起的相关疾病;
7)治疗HBV引起的相关疾病;
8)进行HBV相关的基础科学研究。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(一)本发明提供的HBV纳米抗体,有效克服目前HBV抗体种类少、稳定较差、生产成本高、临床效果不理想等缺点,具有体积小、亲和力高、稳定性强等优点,并且可以低成本、大批量快速生产。
(二)本发明提供的纳米抗体不仅可以作为初期感染阻断、早期感染诊断、中晚期感染治疗的潜在药物,还可以用于体内成像和体外快速检测,例如生产生物活体成像造影剂、ELISA检测/诊断试剂盒、胶体金检测/诊断试剂盒。
(三)基于本发明抗体建立的ELISA检测方法能准确灵敏地检测样品中是否含有HBV。样品的前处理过程简单,耗时少,能同时检测大量的样品,样品检测成本远低于传统的核酸检测方法。
(四)将本发明抗体应用于胶体金检测/诊断试剂盒可以迅速且准确检测样品中是否含有HBV,为解决HBV感染的快速现场诊断提供有力工具。
(五)将本发明抗体应用于乙肝相关基础科学研究可以大幅提高实验灵敏度和精确性,有望解决常规抗体自身无法克服的缺陷如体积大、稳定性差等导致的技术难题。
附图说明
图1为本发明较佳实施例中纳米抗体与HBV ADR亚型小S蛋白的结合曲线。
图2为本发明较佳实施例中纳米抗体与HBV ADR亚型小S蛋白的亲和力曲线(以抗体A1为例)。
图3为本发明较佳实施例中所述抗体的序列及其CDR区。
图4为本发明中所使用的pComb3Xss的质粒图谱。
具体实施方式
本发明旨在提供一种针对HBV的高亲和力抗体,该抗体可有效检测HBV,并对其具有潜在的阻断和治疗作用。
具体地,本发明涉及与乙肝病毒表面小S蛋白特异性结合的骆驼源纳米抗体及其抗原结合片段,具体涉及能以高亲和力结合HBV ADR亚型和ADW亚型表面小S蛋白的骆驼源纳米抗体或其抗原结合片段,其能够用于检测、诊断由HBV引起的感染,并且为预防和治疗由HBV感染引起的肝炎、肝癌及其并发症打下坚实基础。
在本发明的具体实施方案中,提供了抗HBV纳米抗体,其可以与HBV的包膜S蛋白结合,亲和力达到了纳摩尔级别。
在本发明的具体实施方案中,可以进行多种基于抗原/抗体反应的酶联免疫分析检测方法的建立和检测产品的开发。
在本发明的具体实施方案中,可以进行同种或多种基于纳米抗体的多价化等基因工程改造。
在本发明的具体实施方案中,提供了针对HBV的纳米抗体,所述纳米抗体包含如下的氨基酸序列和功能特性:
i)SEQ ID NO:1-8所示的氨基酸序列;或者所述抗体可具有SEQ ID NO:9-15任一所示的高度可变区CDR1氨基酸序列;SEQ ID NO:16-22任一所示的高度可变区CDR2氨基酸序列;和SEQ ID NO:23-30任一所示的高度可变区CDR3氨基酸序列;
ii)所述纳米抗体与HBV具有纳摩尔级别的亲和力。
本发明还提供一种含有所述编码所述抗体的核酸分子的生物材料,所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。
本发明还提供所述抗体的以下任一应用:
1)用于HBV相关的科学研究;
2)用于HBV包膜S蛋白的检测;
3)用于研制HBV检测试剂或ELISA检测试剂。
本发明中,在进行分析检测时,向包被有HBV表面抗原的酶标板的各孔中加入不同浓度所述纳米抗体,由于每个孔中的固相抗原含量均一致,因此当结合在固相抗原上的抗体少,加入的酶标二抗与被结合的纳米抗体结合量少,最后加入底物液和显色液,显色反应浅,用酶标仪检测的OD值低;反之,当所述纳米抗体和固相抗原结合多时,则所测的OD值高,根据加入的纳米抗体量和对应孔的OD值绘制纳米抗体和HBV的结合曲线。
具体地,本发明采用如下技术方案:
本发明提供一种抗体或其抗原结合片段,其氨基酸序列包含由SEQ ID NO:9-15任一所示的CDR1、由SEQ ID NO:16-22任一所示的CDR2、由SEQ ID NO:23-30任一所示的CDR3。
优选地,所述抗原结合片段例如为Fv、Fab、Fab'、scFv、F(ab')2、多价化或多特异片段。
进一步地,其氨基酸序列如SEQ ID NO:1-8任一所示;
或者所述抗体或抗原结合片段是包含将SEQ ID NO:1-8任一所示序列自N末端起第1~126位氨基酸进行截短所获得的序列的抗体,或者将SEQ ID NO:1-8任一所示序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的抗体或抗原结合片段。
本发明提供一种基因工程抗体,其包含所述的抗体或抗原结合片段;优选地,所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。
本发明提供一种融合蛋白,其包含所述的抗体或抗原结合片段或所述的基因工程抗体;优选地,融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。
本发明提供一种偶联物,其包含所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白;优选地,所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP,或其组合。
本发明提供一种核酸分子,其编码所述的抗体或抗原结合片段、如所述的基因工程抗体、如所述的融合蛋白或所述的偶联物,其中所述核酸分子为RNA、DNA或cDNA。
本发明提供一种表达载体,其包含所述的核酸分子;
任选地,所述表达载体可以是DNA、RNA、病毒载体、质粒、表达盒、转座子、其他基因转移系统、或其组合;
优选地,所述表达载体包括病毒载体,如噬菌体载体、慢病毒、腺病毒、AAV病毒、逆转录病毒、其他蛋白表达系统、或其组合。
本发明提供一种宿主细胞,其包含所述的表达载体;其中,所述宿主细胞是用于表达外源蛋白的宿主细胞,例如原核表达细胞、真核表达细胞、转基因细胞系;优选地,所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞、植物细胞、动物细胞。
本发明提供一种组织样本或培养物,其通过培养所述的宿主细胞获得。
本发明提供一种蛋白或抗原结合片段,其从所述的组织样本或培养物中分离获得。
本发明提供一种制备所述抗体或抗原结合片段、所述基因工程抗体、所述融合蛋白或所述偶联物或从所述生物材料中获得的培养物的方法,包括从所述的组织样本或培养物中分离/回收目的蛋白或多肽。
本发明提供一种药物组合物,其包含所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物作为活性成分;例如,所述药物组合物为粘膜或表皮外用型药物、皮下注射型药物、血管输入型药物、或其组合;优选地,所述药物还包括药用赋形剂或载体。
本发明提供所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物在制备或从所述生物材料中获得的培养物用于预防、治疗和/或诊断HBV感染的产品或药物中的用途。
本发明提供所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物在制备用于以下功能的产品中的应用:
1)检测HBV抗原;
2)阻断HBV感染;
3)消杀HBV颗粒;
4)诊断HBV引起的相关疾病;
5)治疗HBV引起的相关疾病;
6)进行HBV相关的基础科学研究。
在本发明的具体实施方案中,所述HBV包括ADR和ADW血清亚型。
在本发明的具体实施方案中,所述标签多肽中所包含纯化标签、检测标签、鉴定标签、偶联标签、功能验证标签等功能多肽,例如His标签、HA标签、Flag标签、c-Myc标签、Avi标签等。
在本发明的具体实施方案中,所述融合蛋白中所包含的检测蛋白包括荧光蛋白、荧光素标记蛋白、过氧化物酶等功能蛋白,例如FPs蛋白、HRP蛋白、Alexa Fluor标记蛋白、FITC标记蛋白等。
在本发明的具体实施方案中,所述融合蛋白中所包含的辅助蛋白为用于辅助折叠、辅助表达、辅助溶解、屏蔽毒性蛋白等功能的蛋白,例如GST蛋白、MBP蛋白、SUMO蛋白、NusA蛋白。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
根据本发明的一些优选实施例,所述纳米抗体可以按如下方法进行制备:将HBV表面小S蛋白作为免疫原免疫实验动物骆驼,提取外周血淋巴细胞的总RNA,经反转录及巢式PCR,克隆出纳米抗体重链(VHH)基因片段,通过酶切连接,将基因片段克隆至噬菌粒载体,高效电转化至大肠杆菌,经辅助噬菌体拯救,构建得到噬菌体纳米抗体库,筛选出HBV纳米抗体,将其进行表达纯化,得到灵敏度高的HBV纳米抗体,并且与流行的突变毒株有较高的交叉反应。制备的纳米抗体分子小,可溶性强,耐高温,易纯化,易表达。
根据本发明的一些优选实施例,所述HBV ADR亚型表面小S蛋白作为免疫原和包被抗原HBV均购自上海普欣生物技术有限公司。
所述酶标板为96孔酶标板,包被抗原的包被浓度为1ug/mL。
所述酶标记的二抗为辣根过氧化物酶标记的抗HA标签抗体,浓度为0.1μg/mL。购自Abcam公司,商品编号:ab1265。
所述显色液A液由过氧化脲1g、柠檬酸10.3g、Na2HPO4·12H2O 35.8g、吐温-20 100μL和蒸馏水1000mL配制而成,pH5。
所述显色液B液由四甲基联苯胺700mg、DMSO 40mL、柠檬酸10.3g和蒸馏水1000mL配制而成,pH值2.4。
所述反应终止液为2M的硫酸液。
实施例1 HBV纳米抗体库的构建
取200ug HBV表面小S蛋白(上海普欣生物技术有限公司,货号671-01)与等体积完全弗氏佐剂混合,充分乳化后注射至骆驼,以后每隔两周加强免疫一次,其中在加强免疫中使用不完全弗氏佐剂与免疫原的混合液,颈背部皮下多点免疫,共免疫5次。从第三次免疫开始,每次免疫后一周从颈锁静脉采血并检测血清效价。
从第5次免疫后的外周血中分离白细胞,提取总RNA,经反转录PCR及巢式PCR,克隆得到VHH基因片段,用限制性内切酶SfiI修饰粘性末端,通过T4连接酶将VHH基因片段连接至噬菌粒pComb3Xss(由University of California,Davis的Bruce D Hammock教授惠赠,结构见图4),高效电转化至大肠杆菌ER2738(购自英国NEB公司),构建HBsAg的噬菌体纳米抗体库。经测定,初级库容量达109cfu,加入辅助噬菌体(感染复数为20:1)M13KO7(购自NEB公司,货号:N0315S)进行拯救,得到噬菌体纳米抗体库,库容量为1012pfu/mL,库的多样性较好。
反转录PCR:
反转录试剂盒采用PrimeScriptTM RT-PCR Kit,购自Takara公司,商品编号:AK2701。
反转录体系如下:
65℃反应5min。取出置于冰上,按以下体系加样,进行cDNA第一链合成。
30℃10min;42℃1h;72℃5min。
巢式PCR:(购自TAKATA公司,货号:6210A)
第一轮PCR:
反应体系如下:
反应程序如下:
第二轮PCR:
反应体系如下:
反应程序如下:
巢式PCR引物序列如下(5′-3′):
GSP-RT:CGCCATCAATRTACCAGTTGA
LP-leader:GTGGTCCTGGCTGCTCTW
R:CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG
F:CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC
其中,R表示碱基A/G,W表示碱基A/T,K表示碱基G/T。
实施例2 HBV纳米抗体的筛选
在96孔酶标板的第1个孔包被HBV表面小S蛋白抗原,包被浓度为1ug/mL,4℃过夜;次日,倒出包被液,用PBST洗涤3次,将酶标板第1、2两个孔用BSA封闭,室温孵育2h;倒出封闭液,用PBST洗涤3次;将实施例1获得的噬菌体纳米抗体库加入第1个孔,反应2h;倒出液体,在洁净的吸水纸上拍干,用PBST洗涤5次;将100μL HBV S蛋白抗原添加到第1个孔中,反应1h;吸出第1个孔中的液体,加入第2个孔,反应1h,除去与BSA结合的噬菌体;收集洗脱液,取5μL用于滴度测定,其余用于扩增。
将噬菌体洗脱液加入新鲜的大肠杆菌ER2738菌液(购自NEB公司),37℃,静置15min;加入羧苄青霉素和SB培养基,37℃,220rpm,培养2h;加入辅助噬菌体M13KO7(感染复数MOI=20:1)(购自NEB公司,货号:N0315S)和卡那霉素,培养过夜;次日,离心取上清,加入PEG-NaCl溶液沉淀纯化噬菌体。
将扩增产物进行下一轮筛选,保证每轮筛选的加入量相同,抗原包被浓度及S蛋白竞争洗脱浓度按2倍递减,计算每轮的滴度,挑取单克隆进行扩增及ELISA鉴定。经3轮淘选得到阳性单克隆。
实施例3 HBV纳米抗体的表达
提取阳性单克隆质粒,转化至大肠杆菌TOP10F’感受态细胞(购自ThermoFishier),复苏后涂布于固体培养基过夜培养。次日,挑取单个克隆于SB-羧苄培养基中培养,加入IPTG诱导过夜表达;次日,用高压均浆仪裂解细胞,滤膜过滤后用镍柱纯化,即利用组氨酸标签与镍柱中氯化镍的亲和层析对纳米抗体进行分离纯化,得到高纯度的抗HBV纳米抗体,即抗体A1-A8,经氨基酸测序分析,所得纳米抗体的氨基酸序列如SEQ ID NO:1-8所示,抗体的CDR区见图3。
实施例4纳米抗体与HBV S蛋白的结合曲线
将HBV ADR亚型表面小S蛋白(上海普欣生物技术有限公司)分别包被于96孔酶标板上,每个孔包被浓度为1ug/mL,4℃过夜反应;次日,甩出孔中的液体,用含0.05%吐温的PBST洗3次,将酶标板倒置在吸水纸上拍干;加入封闭液,37℃孵育30分钟,甩出孔中的液体,用0.05%PBST洗3次,将酶标板倒置在吸水纸上拍干;分别加入100μL不同稀释倍数的实施例3所得的纳米抗体液,37℃孵育30分钟;甩出孔中的液体,用PBST洗3次,将酶标板倒置在吸水纸上拍干;加入酶标二抗(辣根过氧化物酶标记的抗HA标签抗体,购自Roche公司),37℃孵育30分钟;甩出孔中的液体,用PBST洗板3次,拍干;取A液和B液等体积混匀,每孔加100μL,避光显色10~15分钟,加入终止液终止反应,酶标仪上测定各孔在波长为450nm处的OD值。根据抗体浓度和对应孔中的OD值绘制纳米抗体和HBV S蛋白的结合曲线(图1)。
实施例5纳米抗体与HBV表面小S蛋白的亲和力曲线
亲和力检测使用的是亲和素探针,利用Octec red 96仪器进行检测,所述亲和力检测方法是本领域常规技术操作,具体操作如下。向黑色无结合力的96孔板第一列的8个孔中加入0.02%吐温-20的PBST;再向第二列8个孔中加入浓度为15ug/ml生物素标记的HBVADR亚型表面小S蛋白第三、五、七、九、十一列中加入PBST,第四、六、八、十列中加入倍比稀释的本发明所述的纳米抗体,其中每列的第8个孔加入PBST,第十二列中加入甘氨酸2.0,所述上述液体均200ul每孔。主要程序如下:
1)先将8根亲和素探针(streptavidin-sensor,购自FORTEBIO,货号:18-5019)浸入到第一列PBST中进行平衡60s;
2)再将亲和素探针浸入到HBV表面小S蛋白稀释液中结合3min;
3)再回到第一,三列PBST中进行两次平衡;
4)平衡后的探针浸入到第四列纳米抗体稀释液中,进行抗原抗体的特异性结合,结合3min;
5)再回到第三列PBST中进行解离,解离10min。
6)解离后探针在第十二列甘氨酸2.0中再生5s,将结合的纳米抗体完全洗脱下来;
7)再回到第十一列PBST中进行中和5s;
8)重复步骤6)、7);
9)再将探针浸入第五列PBST中进行平衡;重复步骤4)~8)依次检测其他纳米抗体与HBV S蛋白结合能力;
10)最后将实验数据导入到excel表中;
结果参见图2和表1,结果显示八个纳米抗体对HBV ADR亚型表面小S蛋白(ADR-S)的亲和力范围为8.51-9.84nM。
表1纳米抗体与HBVADR亚型表面小S蛋白的亲和力常数KD(M)
ADR-S |
A1 8.69e-9 |
A2 8.51e-9 |
A3 8.98e-9 |
A4 9.61e-9 |
A5 9.84e-9 |
A6 8.72e-9 |
A7 9.44e-9 |
A8 9.39e-9 |
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
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Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Asp Trp Ala Gly Arg Lys Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Ala Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Ser Ile Trp Tyr Thr Ser Gly Ser Asp Tyr Val Pro
100 105 110
Ser Tyr Phe Arg Gly Ala Gly Thr Gln Val Thr Val Ser Ala
115 120 125
<210> 8
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Thr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Thr Thr Trp Ser Gly Arg Lys Thr Asp Tyr Ala Asp Val Val
50 55 60
Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Ser Ile Trp Tyr Thr Gly Gly Ser Asp Tyr Val Pro
100 105 110
Ser Tyr Phe Arg Gly Ala Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gly Phe Ala Phe Ser Ser Tyr Thr
1 5
<210> 10
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ala Phe Ala Phe Ser Ala Tyr Thr
1 5
<210> 11
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Phe Ala Phe Ser Ala Tyr Thr
1 5
<210> 12
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Thr Ala Phe Ser Ala Tyr Thr
1 5
<210> 13
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gly Phe Ala Phe Ser Ser Tyr Ala
1 5
<210> 14
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Ala Phe Ala Ala Ser Ala Tyr Thr
1 5
<210> 15
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ala Phe Ala Phe Ser Ser Tyr Ala
1 5
<210> 16
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ile Asp Val Gly Gly Ser Lys Thr
1 5
<210> 17
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Thr Asp Trp Ser Gly Arg Lys Thr
1 5
<210> 18
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Ile Asp Trp Ser Gly Ser Lys Thr
1 5
<210> 19
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Thr Asp Val Ser Gly Ser Lys Thr
1 5
<210> 20
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Thr Asp Trp Gly Gly Ser Lys Thr
1 5
<210> 21
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Ile Asp Trp Ala Gly Arg Lys Thr
1 5
<210> 22
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Thr Thr Trp Ser Gly Arg Lys Thr
1 5
<210> 23
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Ala Lys Asp Pro Ser Ile Trp Tyr Thr Ser Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Tyr
<210> 24
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Lys Asp Pro Ser Ile Trp Tyr Thr Ala Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Thr
<210> 25
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Ala Lys Asp Pro Ser Ile Trp Tyr Thr Ser Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Phe
<210> 26
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Ala Asp Pro Ser Ile Trp Tyr Thr Gly Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Phe
<210> 27
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Ala Lys Asp Pro Ser Ile Val Tyr Thr Ser Gly Ser Asp Trp Gly Pro
1 5 10 15
Ser Tyr Phe
<210> 28
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Ala Lys Ala Pro Ser Ile Trp Gly Thr Ser Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Thr
<210> 29
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Ala Ala Asp Pro Ser Ile Trp Tyr Thr Ser Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Phe
<210> 30
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ala Lys Asp Pro Ser Ile Trp Tyr Thr Gly Gly Ser Asp Tyr Val Pro
1 5 10 15
Ser Tyr Phe
Claims (10)
1.乙肝病毒表面S蛋白高亲和力纳米抗体或其抗原结合片段,其特征在于,其高变区CDR1、CDR2和CDR3分别选自SEQ ID NO:9-15、SEQ ID NO:16-22和SEQ ID NO:23-30任一所示的氨基酸序列;
所述抗原结合片段为Fv、Fab、Fab'、scFv、F(ab')2、多价化或多特异片段。
2.根据权利要求1所述的纳米抗体或其抗原结合片段,其特征在于,其氨基酸序列如SEQ ID NO:1-8任一所示;或者
SEQ ID NO:1-8任一所示序列经截短得到的氨基酸序列;或者,
将SEQ ID NO:1-8任一所示序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的抗体或抗原结合片段。
3.基因工程抗体,其特征在于,其包含权利要求1或2所述的抗体或其抗原结合片段;
其中,所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。
4.融合蛋白,其特征在于,其包含权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体;
其中,所述融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。
5.偶联物,其特征在于,其包含权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白;
其中,所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP,或其组合。
6.核酸分子,其特征在于,其编码权利要求1或2所述的抗体或其抗原结合片段,或编码权利要求3所述的基因工程抗体,或编码权利要求4所述的融合蛋白,或编码权利要求5所述的偶联物;
其中,所述核酸分子为RNA、DNA或cDNA。
7.含有权利要求6所述核酸分子的生物材料,其特征在于,所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
8.药物组合物,其特征在于,活性成分为权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物或从权利要求7所述生物材料中获得的培养物;
所述药物组合物为外用型、皮下注射型、血管输入型,或其组合;
优选地,所述药物组合物还包括药用赋形剂或载体。
9.权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物或从权利要求7所述生物材料中获得的培养物在制备用于预防、治疗和/或诊断HBV感染、相关科学研究的生物制品中的应用。
10.权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物或从权利要求7所述生物材料中获得的培养物的以下任一应用:
1)用于制备预防或治疗由乙肝病毒感染以及由其感染所致相关疾病的药物;
2)用于制备乙肝病毒检测试剂或试剂盒;
3)用于进行乙肝相关的基础科学研究。
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