CN116925213A - 一种中和a型肉毒毒素的纳米抗体 - Google Patents
一种中和a型肉毒毒素的纳米抗体 Download PDFInfo
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- CN116925213A CN116925213A CN202311169052.7A CN202311169052A CN116925213A CN 116925213 A CN116925213 A CN 116925213A CN 202311169052 A CN202311169052 A CN 202311169052A CN 116925213 A CN116925213 A CN 116925213A
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Abstract
本发明公开了一种中和A型肉毒毒素的纳米抗体,所述纳米抗体的CDR1的氨基酸序列如SEQ ID NO.2所示,所述CDR2的氨基酸序列如SEQ ID NO.3所示,所述CDR3的氨基酸序列是GEF。本发明的实验证明,该纳米抗体与BoNT/A抗原具有较强亲和力,施用该纳米抗体可以治疗BoNT/A引发的肉毒毒素中毒。因此本发明为临床诊断或治疗肉毒毒素中毒提供了新的途径。
Description
技术领域
本发明属于生物医药领域,涉及一种中和A型肉毒毒素的纳米抗体。
背景技术
肉毒毒素又被称为肉毒神经毒素,是由厌氧的革兰氏阳性菌肉毒梭菌产生的一种毒素蛋白,是已知最强的毒素,根据其抗原性不同可分为七种类型(BoNT/A-BoNT/G),其中A型肉毒毒素中毒致死率最高。在小鼠中测得BoNTs的腹腔注射半数致死量(LD50)为0.5-5ng/kg,在人体内的LD50大约为1 ng/kg。肉毒毒素中毒的潜伏期一般为数小时到数天,中毒者一般在2-3 d死亡。中毒后可引起虚弱、眩晕、视物不清、骨骼肌麻痹、严重时引起呼吸衰竭导致死亡。在日常生活中,由于食物污染肉毒毒素和美容行业不规范注射肉毒毒素导致的中毒事件时有发生,严重威胁人们的生命健康。因此,对于预防和治疗肉毒毒素中毒的研究具有十分重要的意义。
目前,临床上肉毒毒素中毒的主要治疗方法有两种:特异性的马血清抗毒素治疗和对症治疗。在预防肉毒毒素的主要策略是接种疫苗,自二战以来美国已经研制多种针对肉毒毒素疫苗,AB型二价类毒素疫苗和PBT类毒素疫苗等,用于高危人群的接种。但是类毒素疫苗仍存在较多的缺陷,PBT一直未获得FDA批准上市。因此获得有效防治肉毒毒素中毒的特效药,对于生物安全具有重要意义。而治疗性抗体因其高特异性、低毒副作用、良好的中和效应,成为BoNT/A特效解毒剂研发团队的首选和研究重点。
纳米抗体是目前已知最小的活性抗原结合蛋白,1993年由Hamers团队在骆驼的血清中发现了一种仅由两条重链构成的新型抗体即重链抗体,而重链抗体的可变区片段就是纳米抗体。与常规IgG抗体相比,纳米抗体的优势在于其结构稳定、特异性强、产量高、易于表达,作为研究工具、诊断和治疗方法受到广泛关注。此外,在抗原识别过程中,由于纳米抗体有着较长的互补决定区,易于形成loop环结构与蛋白质空间构象上的裂隙和空腔结合,从而识别隐匿的凹型表位如酶活性位点和隐匿的病毒表位。因此在肉毒毒素治疗药物或诊断试剂的研发中,纳米抗体是良好的候选药物。
发明内容
本发明公开了一种A型肉毒毒素的纳米抗体,所述纳米抗体包含SEQ ID NO.1所示的重链可变区的CDR1、CDR2和CDR3。
进一步,所述CDR1的氨基酸序列如SEQ ID NO.2所示,所述CDR2的氨基酸序列如SEQ ID NO.3所示,所述CDR3的氨基酸序列是GEF。
在本发明的具体实施方式中,纳米抗体还包括骨架区;其重链可变区的结构为:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
在一种可选的实施方式中,纳米抗体为单价纳米抗体、多价纳米抗体、多特异性抗体和融合型纳米抗体中的至少一种。
单价纳米抗体:是用特异性的抗原从纳米抗体库中筛选得到的抗原特异性的纳米抗体,因其表面有大量的亲水残基,能保持严格的单体结构,且仅以这种单体形式就能高特异性、高亲和力地与其抗原相结合。
多价纳米抗体:多价抗体是识别同一种表位的单价抗体的聚合物,比对应的单价纳米抗体具有更高的抗原亲和力。多特异性抗体是识别不同表位的单价抗体的聚合物,能结合不同靶目标或同一靶目标的不同表位,比单价抗体具有更高的抗原识别能力。而纳米抗体结构简单,只有一个结构域,可通过短小的连接序列聚合在一起,从而转换成多价和多特异性的形式。
融合型纳米抗体:纳米抗体具有严格的单体特点且其相对分子质量很小,很容易通过基因工程技术与其他结构(如BSA、IgG-Fc等)结合形成新的融合分子,如能延长其半衰期的酶、抗菌肽或显影物质等。在新的融合分子中,纳米抗体与其靶抗原定向结合,与纳米抗体融合的部分就能发挥相应的功能。在临床,医生希望药物能在体内停留足够长的时间,然而纳米抗体的血液清除速度却很快,不利于它所携带的药物发挥作用。所以,通过基因技术将纳米抗体VHH和寿命较长的分子融合在一起,可以提高纳米抗体在血液中的存在时间即延长其半衰期,从而达到更好的治疗效果。
在本发明的一个具体实施例中,所述纳米抗体是单价纳米抗体。
进一步,所述纳米抗体的氨基酸序列如SEQ ID NO.1所示。
本发明还公开了一种核酸分子或包含所述核酸分子的重组载体,所述核酸分子编码前面所述的纳米抗体。
在一种可选的实施方式中,所述核酸分子的核苷酸序列如SEQ ID NO.8所示。
考虑到密码子的简并性,可在其编码区,在不改变氨基酸序列的条件下,编码上述抗体的基因序列可以进行修改,获得编码相同抗体的基因;也可以根据表达抗体宿主的密码子偏爱性,人工合成改造基因,以提高抗体的表达效率。
重组载体为表达载体或克隆载体,优选为表达载体,可以指任何重组的多核苷酸构建体,该构建体可通过转化,转染或转导的方式将目的DNA片段直接或间接(如包装成病毒)导入宿主细胞内,进行目的基因表达。
其中一种类型的载体是质粒,即环状双链DNA分子,可将目的DNA片段连接至质粒环中。另一种类型的载体为病毒载体,其可将目的DNA片段连接包装至病毒基因组中(如腺病毒,腺相关病毒,逆转录病毒,慢病毒,溶瘤病毒)。这些载体进入宿主细胞后,可以进行目的基因的表达。
本发明还公开了一种宿主细胞,其包含前面所述的核酸分子或前面所述的重组载体。
宿主细胞例如选自哺乳动物细胞,哺乳动物细胞选自293细胞、293T细胞、293FT细胞、CHO细胞、COS细胞、小鼠L细胞、LNCaP细胞、633细胞、Vero、BHK细胞、CV1细胞、Hela细胞、MDCK细胞、Hep-2细胞和Per6细胞中的任一种。其中的293系列细胞、Per6细胞和CHO细胞是用于生产制备抗体或重组蛋白的常用哺乳动物细胞,为本领域普通技术人员所熟知。
本发明还公开了一种检测、富集或纯化BoNT/A的产品,所述产品包含前面所述的纳米抗体。
所述产品包括选自试剂、试剂盒或芯片。
进一步,所述试剂是抗体,其含有前面所述的纳米抗体。
在一些实施例中,抗体可以为全长抗体、重链抗体、嵌合抗体、多特异性抗体(例如双特异性抗体、三特异性抗体、四特异性抗体等等)、鼠源抗体、人源化抗体或抗原结合片段中的任意一种。抗原结合片段包括所述抗原结合片段选自抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种,只要它们展示出所期望的抗原结合活性。
本发明所述的“嵌合抗体”是将非人源性抗体的可变区与人抗体的恒定区或骨架区融合而成的抗体,可以减轻非人源性抗体诱发的免疫应答反应。
上述抗原结合片段也即抗体的功能性片段,通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本发明记载的内容容易理解到,上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原***二硫键的方法获得。在本发明公开了完整抗体的结构基础上,本领域技术人员容易获得上述的功能性片段。
上述抗原结合片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪,比如Applied BioSystems等销售的自动肽合成仪合成获得。
进一步,所述试剂是纳米颗粒,其含有前面所述的纳米抗体或其前面所述的抗体。
纳米颗粒选自有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒中的任意一种。
进一步,所述试剂盒包含前面所述的纳米抗体、前面所述的抗体或前面所述的纳米颗粒。
在一种实施方式中,包被有纳米抗体或抗体的纳米颗粒作为捕获抗体,标记有可被检测的标记物的纳米抗体或抗体作为检测抗体。
在另外一种实施方式中,当试剂盒仅包括:标记有可被检测的标记物的纳米抗体或抗体;此时的纳米抗体或抗体可用于捕获或检测抗体。
进一步,可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和胶体中的至少一种。
荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中,催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中,放射性同位素包括但不限于212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu和18F。
在一种可选的实施方式中,化学发光试剂选自吖啶酯、鲁米诺、光泽精、甲壳动物荧光素、联吡啶钌、二氧环乙烷、洛粉碱和过氧草酸盐中的至少一种。
胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中,胶体金属包括但不限于胶体金、胶体银和胶体硒。
在一种可选的实施方式中,富集或纯化BoNT/A的产品包括载体以及载体上的纳米抗体或抗体。
在一种可选的实施方式中,载体选自磁珠、琼脂糖凝胶微球、硅胶微球或多孔材料。例如将活化后的磁珠与抗体进行孵育,使得磁珠上包被抗体。
本发明还公开了一种检测、富集或纯化BoNT/A的方法,所述方法包括将包含BoNT/A的样品与前面所述的纳米抗体或前面所述的抗体接触。
本发明还公开了前面所述的纳米抗体或前面所述的抗体在制备检测、富集或纯化BoNT/A的产品中的应用。
本发明还公开了前面所述的纳米抗体或前面所述的抗体在制备诊断A型肉毒毒素引发的肉毒毒素中毒的产品中的应用。
本发明还公开了前面所述的纳米抗体或前面所述的抗体在制备治疗A型肉毒毒素引发的肉毒毒素中毒的药物中的应用。
进一步,所述药物组合物还包括药学上可接受的载体或辅料。
“药学上可接受的”是指当药物适当地给予动物或人时,它们不会产生不利的、过敏的或其它不良反应。
“药学上可接受的载体或辅料”应当与所述有效成分相容,即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如Tween;润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1显示纳米抗体库DNA钓取电泳图,其中,M代表核酸Marker,泳道1为所有免疫球蛋白重链(VH,VHH)的可变区结构域,泳道2为VHH的扩增;
图2显示部分ELISA筛选结果;
图3显示SDS-PAGE结果图;
图4显示SEC-HPLC分析结果图,其中,A:218nm;B:280nm;
图5显示Biacore分析结果图;
图6显示小鼠体内攻毒保护实验结果图。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另外指明,否则实践本发明将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:ALaboratoryManual)》,第二版(Sambrook等人,1989);《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编,1984);《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编,1987);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《哺乳动物细胞用基因转移载体(Gene Transfer Vectors forMammalian Cells)》(J.M.Miller和M.P.Calos编,1987);《当代分子生物学方法(CurrentProtocols inMolecular Biology)》(F.M.Ausubel等人编,1987);《PCR:聚合酶链反应(PCR:ThePolymerase Chain Reaction)》(Mullis等人编,1994);以及《当代免疫学方法(CurrentProtocols in Immunology)》(J.E.Coligan等人编,1991),所述文献中的每个文献均通过引用明确并入本文中。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例一 天然骆驼VHH文库的获取
一、材料
骆驼L9的外周血 100 mL;骆驼淋巴细胞分离液为天津市灏洋生物制品科技有限责任公司产品;RNA Easy Fast动物组织/细胞总RNA提取试剂盒和FastKing cDNA 第一链合成试剂盒为天根生化科技有限公司产品;PCR Primer Max酶为TAKARA公司产品;基因测序由苏州金唯智生物科技有限公司完成;其它相关试剂均为市购。
二、方法和结果
1、骆驼L9外周血的获取
采用EDTA涂层的采血管从骆驼颈静脉收集100 mL血液,倒置两次防止凝血,将血液样本转移到实验室。
2、淋巴细胞的分离
采用15 ml离心管,按照分离液5 mL,血液5 mL,室温600 g离心25 min。离心后,离心管由上至下分为四层。第一层为血浆层,第二层为环状乳白色骆驼淋巴细胞层,第三层为透明分离液层,第四层为红细胞层。小心吸取骆驼淋巴细胞,加入新的50 mL离心管中,加入清洗液,250 g离心15 min,弃去上清。重复清洗两遍,重悬所得细胞为骆驼淋巴细胞。
3、天然VHH文库的调取
取上述淋巴细胞6×107按照RNA Easy Fast动物组织/细胞总RNA提取试剂盒说明书操作获得总RNA,按照FastKing cDNA 第一链合成试剂盒说明书,获得cDNA文库。
利用引物CALL001和CALL002扩增来自cDNA的所有免疫球蛋白重链(VH,VHH)的可变区结构域,经琼脂糖凝胶电泳,约1000bp的片段代表常规重链抗体,约750bp的片段代表仅重链抗体即纳米抗体;胶回收750bp处的片段,获得纯化的DNA片段。利用引物VHH-Back和VHH-For进行巢式PCR,最终获得纳米抗体的基因文库(图1)。
引物如下:
CALL001:5’-GTCCTGGCTGCTCTTCTACAAGG-3’ (SEQ ID NO.9);
CALL002:5’-GGTACGTGCTGTTGAACTGTTCC-3’ (SEQ ID NO.10);
VHH-Back:5’- CCGTGGCCCAGGCGGCCGTCCTGGCTGCTCTTCTACA -3(SEQ ID NO.11)’;
VHH-For:5’-TGCTGGCCGGCCTGGCCTGAGGAGAYGGTGACCWGGGT -3’ (SEQ ID NO.12)
实施例二 天然骆驼VHH噬菌体文库的构建
一、材料
天然纳米抗体的基因文库;PCR Primer Max酶为TAKARA公司产品;T4 DNA连接酶为NEB公司产品;基因测序由苏州金唯智生物科技有限公司完成;其它相关试剂均为市购。
二、方法和结果
1、纳米抗体库构建
a)纳米抗体文库片段酶切,连接pComb3XTT载体:
纳米抗体库PCR产物 10 μL
Shif I 1 μL
Cutstmart 5 μL
无菌水补足总体积为 50 μL
上述体系在37℃孵育6 h,经普通DNA产物纯化试剂盒进行回收。
b) 上述酶切产物,连接pComb3XTT载体:
纳米抗体库PCR产物 1 μL
pComb3XTT载体 5 μL
T4连接酶 1 μL
T4 Buffer 1 μL
无菌水补足总体积为 10 μL
16℃连接过夜,连接产物电转化TG1大肠杆菌,涂布在含有100 μg/mL氨卞青霉素(终浓度)和2%葡萄糖的2YT琼脂培养基上。同时进行梯度稀释,获得滴定板计算库容量,挑取单克隆菌落进行测序。获得的噬菌体纳米抗体库菌在含有100 μg/mL氨卞青霉素(终浓度)的2%葡萄糖的2YT液体培养基中培养过夜,用PEG沉淀的方法获得噬菌体纳米抗体库。
纳米抗体文库经过酶切,酶连等分子生物学技术构建至噬菌体载体上,噬菌体纳米抗体库菌落经过测序鉴定多序列比对,鉴定库质量;噬菌体库经扩增、PEG沉淀,成功获得噬菌体纳米抗体库,用于后续筛选。
实施例三 抗BoNT/A纳米抗体的噬菌体天然纳米抗体库筛选
一、材料
噬菌体天然纳米抗体库;BoNT/A AHC抗原;PBS;Tween-20;TG1;辣根酶标记的抗M13抗体;其他试剂为市购产品。
二、方法
1、淘洗
1.1 10 μg/mL无菌PBS稀释BoNT/AHC 抗原包被免疫管,500 μL每管,4℃包被过夜(抗原总量为5 μg)。
1.2 弃去抗原溶液,加入无菌PBST配置的4%牛奶,室温封闭1 h。同时准备500 μL4%牛奶稀释的噬菌体抗体库5×1012cfu室温孵育1 h。
1.3 在封闭的同时,接种200 μL TG1至50 mL 2YT液体培养基中,培养至OD600为0.8。
1.4 弃去牛奶,在免疫管中加入500 μL 封闭后的噬菌体溶液,室温孵育1 h。
1.5 PBST洗管15次,然后用PBS洗管10次。
1.6 500 μL 0.1 M HCL-Gly洗脱噬菌体,室温洗脱10 min(中间轻微晃动以检查)。加入100 μL 1 M Tris-HCl,以调整洗脱液pH值至pH 7.5。
1.7 再次使用500 μL 0.1 M HCL-Gly洗脱噬菌体,室温10 min,将二次洗脱液加入第一次洗脱液和Tris的混合溶液中。
1.8 将上述获得的噬菌体取500 μL加入10 mL对数生长期的TG1中,37℃静置培养0.5 h。
1.9 4000 rpm 20℃离心15 min。
1.10 弃去上清,将所有沉淀涂布于2YTAG(2YT+Amp 100 μg/mL+ 2%葡萄糖)平板(15 cm大平板)上,37℃培养过夜。
1.11 将所有TG1菌株从平板上刮下后加入至50 mL 2YTAG(2YT+Amp 100 μg/mL +2%葡萄糖)培养中,37℃,220 rpm培养2.5 h使OD值达到约0.1。
1.12菌株生长到达对数期后取10 mL,在其中加入20~200 μL M13KO7辅助噬菌体,37℃静置培养0.5h。
1.13 4000 rpm 20℃离心15 min。
1.14 弃去上清,50 mL 2YTAK(2YT+Amp 100 μg/mL+Kana 70 μg/mL)培养基重悬,28℃震荡培养过夜。
1.15 如上所述沉淀噬菌体进行下一轮淘洗,共进行3轮淘洗,富集挑取单克隆进行ELISA验证。
2、ELISA方法分析单克隆噬菌体
2.1 在96深孔板中生产单克隆噬菌体
2.1.1 40 μL TG1接种于10 mL新鲜 2YT 培养基中,37℃ 220 rpm 培养3 h。
2.1.2 将洗脱后的噬菌体500 μL加入10 mL TG1中,37℃ 孵育0.5 h。
2.1.3 将感染后的菌液十倍倍比稀释(10-1,10-2…10-8)于2YT培养基中,每个稀释样品取50 μL,涂布于2YTAG(2YT+Amp 100 μg/mL)固体培养基上,37℃过夜。
2.1.4 96孔深板中每孔加入900 μL 2YTAG(2YT+Amp 100 μg/mL)培养基,每个平板挑取单克隆,在96孔深板中37℃,220 rpm,培养4 h。
2.1.5 每孔吸出100 μL与96孔板保存,用于后续测序。
2.1.6 每孔加入10倍稀释的M13KO7 100 μL (1.2 mL M13KO7辅助噬菌体+12 mL2YT培养基),37℃静置孵育0.5 h。
2.1.7 96孔深板1800 rmp 4℃离心15 min。
2.1.8 弃去上清,板子置于28℃摇动10 min。
2.1.9 每孔加入1 mL新鲜的2YTAK(2YT+Amp 100 μg/mL + Kana 70 μg/mL)培养基,28℃培养过夜。
2.1.10 96孔深板1800 rmp 4℃离心15 min,获得单克隆噬菌体上清。
2.2 ELISA验证
2.2.1 板条中包被BoNT/A抗原、阴性对照和阳性对照,4℃包被过夜。
2.2.2 每孔加入200 μL PBSM(PBS配置的4%脱脂牛奶)室温封闭1 h。
2.2.3 弃去孔内溶液后每孔加入100 μL 噬菌体,室温孵育1 h。
2.2.4 0.1% Tween-20去离子水配置洗液洗板3次。
2.2.5 每孔加入100 μL anti-M13/HRP抗体(1:5000 稀释),室温孵育45 min。
2.2.6 0.1% Tween-20去离子水配置洗液洗板5次。
2.2.7 每孔加入100 μL TMB显色。
2.2.8 加入中止液后酶联仪测OD450 nm。
部分筛选结果如图2,1号抗体与抗原结合最好,选择1号抗体进行表达并命名为HM。
经测序鉴定,HM抗体的氨基酸序列如SEQ ID NO.1所示,其中 所述CDR1的氨基酸序列如SEQ ID NO.2所示,所述CDR2的氨基酸序列如SEQ ID NO.3所示,所述CDR3的氨基酸序列是GEF。FR1-4的氨基酸序列分别如SEQ ID NO.4-7所示。HM抗体的核苷酸序列如SEQ IDNO.8所示。
SEQ ID NO.1:EVQLVESGGGLVQPGGSLRLSCEASGFGSWFRFDENTVNWYRQPPGKSREFDELVARYPKSGIVTYLDSVKGRFTISRDNAKKMAFLQMNSLKPEDTAVYYCNVGEFWGQGTQVTVSS;
SEQ ID NO.2:
GFGSWFRFDENTVN
SEQ ID NO.3:
LVARYPKSGIVT
CDF3:GEF
SEQ ID NO.4:
EVQLVESGGGLVQPGGSLRLSCEAS
SEQ ID NO.5:
WYRQPPGKSREFDE
SEQ ID NO.6:
YLDSVKGRFTISRDNAKKMAFLQMNSLKPEDTAVYYCNV
SEQ ID NO.7:
WGQGTQVTVSS
SEQ ID NO.8:GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGAGGCAAGTGGGTTTGGAAGCTGGTTTAGGTTTGACGAGAACACCGTGAACTGGTATAGGCAGCCCCCTGGAAAGAGTAGGGAGTTTGACGAGCTGGTGGCAAGGTACCCTAAGAGCGGAATCGTGACCTACCTGGACTCCGTGAAGGGCAGGTTCACCATCTCTAGGGACAACGCAAAGAAGATGGCCTTCTTGCAGATGAACTCCTTGAAACCTGAGGACACAGCCGTGTACTATTGCAACGTGGGCGAATTTTGGGGACAGGGAACACAGGTGACCGTGAGCTCC
实施例四 抗BoNT/A纳米抗体HM的表达及性质评价
一、材料
真核表达载体pFRT-IgG1由本室保存;引物由苏州金唯智生物科技有限公司合成;转染试剂为Invitrogen公司产品,羊抗人IgG、及辣根酶标记的羊抗人IgG购自Thermo;内切酶为NEB公司产品;质粒提取试剂盒购自天根生化科技有限公司;CHO细胞购自ATCC;CD-1小鼠购自维通利华,其他试剂为市购产品。
二、方法
1、抗体的表达
1.1 抗体真核表达载体的构建
pFRT-IgG1是含有人IgG1抗体恒定区基因的高效表达载体。将实施例二所述获得的纳米抗体基因克隆载体用相应的内切酶消化(纳米抗体基因用Nhe I和Xho I消化)后,依次与经相同内切酶消化的载体连接,获得真核表达载体。具体实施如下:
1.1.1 如实施例二所述,获得的纳米抗体HM基因经PCR获得带有酶切位点的目的片段;
1.1.2 取HM PCR产物用Nhe I 和XhoI消化;
1.1.3 取1 μg pFRT-IgG1载体,用Nhe I和XhoI消化。用DNA连接酶T4连接所得的经Nhe I及XhoI消化的pFRT-IgG1载体和用Nhe I及XhoI消化的HM PCR产物。连接产物转化TOP10大肠杆菌,涂布在含有100 μg/mL氨卞青霉素(终浓度)的LB琼脂培养基上。获得的克隆在含有100 μg/mL氨卞青霉素(终浓度)的LB液体培养基中培养,用质粒提取试剂盒(天根生化科技有限公司)提取质粒。所提取的质粒经Nhe I和Xho I消化后,用1%琼脂糖凝胶电泳分析,选择一个携带有抗体HM基因的克隆。
作为上述程序的结果,获得的携带抗BoNT/A纳米源抗体HM基因的质粒pFRT-IgG1-HM。
1.2 纳米抗体HM的表达
将获得的真核表达载体,通过脂质体介导的方法瞬时转染至CHO细胞,8天后收获上清,利用羊抗人IgG以及辣根酶标记的羊抗人IgG进行双夹心ELISA法检测上清中抗体的含量。实验结果显示,上清中目的蛋白可以被羊抗人IgG抗体识别,含有人IgG抗体的恒定区,具有人IgG抗体特征。抗体表达量为770.60mg/L。利用Mabselect Sure亲和层析柱纯化抗体。利用SDS-PAGE电泳检测纯化后抗体,结果如图3所示,表明抗体表达成功。备注:图中M代表蛋白Marker,R代表HM还原条带,N-R代表HM非还原条带。
2、纳米抗体HM的性质鉴定
2.1 SEC-HPLC分析
2.1.1步骤
流动相为50 mmol/mL磷酸钠和200 mmol/mL氯化钠,流速为1 mL/min,进样体积7μL,检测波长为218nm和280nm,采集后采用LabSolutions***对色谱图进行积分并计算相关数据。
2.1.2结果
结果如图4所示,HM主峰保留时间约7.9min,峰面积占比99%,表明HM纯度在不小于99%,统计如表1。
表1、峰图统计图
2.2 Biacore检测纳米抗体的亲和力
2.2.1步骤
a) 开机
Biacore T200按照操作手册进行开机。
b) 样品稀释
将10 HBS-EP+(pH7.4)用去离子水稀释10倍,配制200mL。用运行缓冲溶液稀释抗体HM至0.3μg/mL,样品稀释浓度为10nM,5nM,2.5 nM,1.25 nM,0.625 nM,0.3125nM。
c) 样品检测并分析
运行Kinetics/Affinity程序进行测定并分析结果。
2.2.2结果
结果如图5和表2所示。
表2、纳米抗体亲和力结果
3、纳米抗体HM的功能检测
利用小鼠体内攻毒保护实验研究纳米抗体HM的治疗功能
3.1步骤
选取18-20g CD-1雌性小鼠,设置Control组2倍LD50肉毒毒素,抗体组为不同浓度的抗体与2倍LD50肉毒毒素混合,室温孵育30min,然后腹腔注射200μL/只进行给药。每天观察小鼠2-6次,持续3-5天,观察小鼠状态并记录死亡率。
3.2结果
结果如图6所示,在观察60h后,抗体HM在最低浓度0.25mg/kg时仍能够保护小鼠100%存活,Control组仅存活20%,表明HM能够有效保护小鼠A型肉毒毒素中毒。
上述实施例的说明只是用于理解本发明的方法及其核心思想,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (10)
1.一种中和A型肉毒毒素的纳米抗体,其特征在于,所述纳米抗体包含SEQ ID NO.1所示的重链可变区的CDR1、CDR2和CDR3。
2.根据权利要求1所述的纳米抗体,其特征在于,所述CDR1的氨基酸序列如SEQ IDNO.2所示,所述CDR2的氨基酸序列如SEQ ID NO.3所示,所述CDR3的氨基酸序列是GEF。
3.根据权利要求1所述的纳米抗体,其特征在于,所述纳米抗体的氨基酸序列如SEQ IDNO.1所示。
4.一种核酸分子或包含所述核酸分子的重组载体,其特征在于,所述核酸分子编码权利要求1-3中任一项所述的纳米抗体。
5.一种宿主细胞,其包含权利要求4所述的核酸分子或权利要求4所述的重组载体。
6.一种检测BoNT/A的产品,所述产品包含权利要求1-3中任一项所述的纳米抗体。
7.一种检测、富集或纯化BoNT/A的方法,所述方法包括将包含BoNT/A的样品与权利要求1-3中任一项所述的纳米抗体接触。
8.权利要求1-3中任一项所述的纳米抗体在制备检测、富集或纯化BoNT/A的产品中的应用。
9.权利要求1-3中任一项所述的纳米抗体在制备诊断A型肉毒毒素引发的肉毒毒素中毒的产品中的应用。
10.权利要求1-3中任一项所述的纳米抗体在制备治疗A型肉毒毒素引发的肉毒毒素中毒的药物中的应用。
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