CN114853896A - 一种抗糖基转移酶a亚单位的纳米抗体及其应用 - Google Patents
一种抗糖基转移酶a亚单位的纳米抗体及其应用 Download PDFInfo
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- CN114853896A CN114853896A CN202210421025.3A CN202210421025A CN114853896A CN 114853896 A CN114853896 A CN 114853896A CN 202210421025 A CN202210421025 A CN 202210421025A CN 114853896 A CN114853896 A CN 114853896A
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Abstract
本发明公开了一种抗糖基转移酶A亚单位的纳米抗体及其应用，该纳米抗体可变区具有SEQ ID NO.6所示可变区中的互补决定区CDR‑1、CDR‑2、CDR‑3。本发明提供的抗糖基转移酶A亚单位的纳米抗体具有与糖基转移酶A亚单位特异性结合的活性，并中和糖基转移酶A的活性，可以用于检测艰难梭菌，也可用用于制备治疗抗艰难梭菌的药物；该纳米抗体还具有分子量小、亲和力高、结构和性能稳定等特点；能够耐受胃部酸性环境，不易被胃蛋白酶降解等优势。
Description
本申请为申请号202110077974.X、申请日2021年01月20日、发明名称“一种抗糖基转移酶A亚单位的纳米抗体及其应用”的分案申请。
技术领域
本发明属于抗体技术领域，涉及纳米抗体技术，具体而言，涉及一种抗糖基转移酶A(TcdA)亚单位的纳米抗体及其应用。
背景技术
艰难梭菌(Clostridium difficile，CD)广泛分布于自然环境中，可存活于水，土壤，蔬菜，狗、猫体内及医院环境中，是动物和人肠道中的正常菌群。不规范使用抗生素，使肠道菌群失调，耐药的CD就会大量生长繁殖，导致抗生素相关性腹泻和伪膜性肠炎等疾病，是抗生素相关性腹泻病的主要病原体。CD的芽胞抵抗力强，难消除，可在医院环境长期存在，使CD成为了医院内获得性腹泻的主要病原菌之一。
CD相关疾病的感染与发病主要是产毒型CD在肠道过度增殖并大量释放毒素引起的。正常情况下，肠道中的CD受到双歧杆菌、拟杆菌、优杆菌等优势菌群的抑制，处于劣势，不致病；当长期大量接受广谱抗菌药物、免疫抑制剂或化疗药物治疗后，肠道微生态平衡被打破，肠道菌群失调，CD过度繁殖并释放毒素，引发艰难梭菌感染(Clostridiμm difficileinfection，CDI)相关疾病。
当CD大量繁殖时，其通过菌体或孢子的表面蛋白破坏结肠正常菌群，然后再定殖，在鞭毛和蛋白酶的协助下，进入黏液层，黏附于肠上皮细胞，释放相关毒素，破坏肠细胞、改变细胞骨架、释放粘液和炎症产物，引起肠道感染性疾病。感染后可能出现高热、血水样便、腹胀、中毒性巨结肠等表现。CD能产生6种毒素，主要致病因子为毒素A、毒素B及二元毒素。而其中的毒素A为肠毒素，可与肠黏膜刷状缘细胞上毒素受体结合，趋化白细胞，激活巨噬细胞、肥大细胞及中性粒细胞，释放强效的炎症递质和细胞因子，从而引起局部黏膜血管的通透性增加，绒毛损害，黏膜出血、坏死；在伪膜性肠炎中，毒素A和B最直接的作用是破坏上皮屏障的紧密联接，促进炎症因子的释放、中性粒细胞的移动和伪膜的形成。由于艰难梭菌毒素A和毒素B的C端都具有一个毒素识别和结合细胞受体的功能区，称为受体结合区(receptor-binding domain，RBD)，如果能够研发出针对这一区域的抗体，使RBD被覆盖，就能够直接阻断毒素的结合，使其无法发挥毒素效果，从而达到免疫防御的作用。
由于CD的感染率和感染程度越来越严重，开发新的防治方法就尤为重要了。而鉴于A毒素即糖基转移酶A在CD感染中具有重要的作用，开发能够针对糖基转移酶A的特异性抗体，同样能够在抵抗CDI中发挥主要作用。目前，还没有针对糖基转移酶A RBD的特异性抗体的相关报道。
发明内容
针对目前缺少艰难梭菌有效治疗药物的技术现状，本发明目的旨在提供一种抗糖基转移酶A亚单位(TcdA)的纳米抗体，该纳米抗体具有与糖基转移酶A亚单位的RBD特异性结合的活性，可以用于检测艰难梭菌与抑制艰难梭菌的活性，具有良好的临床应用前景。
本发明提供的抗糖基转移酶A亚单位的纳米抗体，其可变区具有3个互补决定区CDR-1、CDR-2、CDR-3，CDR-1由SEQ ID NO.1所示的氨基酸序列组成，CDR-2由SEQ ID NO.2所示的氨基酸序列组成，CDR-3由SEQ ID NO.3所示的氨基酸序列组成。
在一种优选的实现方式中，所述纳米抗体的可变区序列由SEQ ID NO.4-6所示的氨基酸序列组成。当然本发明的纳米抗体的可变区序列并不限于SEQ ID NO.4-6所示的氨基酸序列，其还可以是在所示序列上经过一个或多个氨基酸残基的替换和/或删除得到的，且具有与SEQ ID NO.4-6相同生物活性(即具有CDR-1、CDR-2、CDR-3三个互补决定区，能够与糖基转移酶A亚单位特异性结合的活性)、或者活性加强、或者活性减弱的衍生序列。这类衍生序列也属于本发明的保护范围。
本发明进一步提供了一种编码上述含有可变区的纳米抗体的核苷酸编码序列，所述编码序列由SEQ ID NO.7、SEQ ID NO.8和SEQ ID NO.9所示。
本发明进一步提供了上述纳米抗体在制备艰难梭菌治疗药物中的应用。
本发明进一步提供了上述纳米抗体在制备艰难梭菌检测试剂中的应用。
与现有技术相比，本发明具有以下有益效果：
1、本发明提供的抗糖基转移酶A亚单位的纳米抗体具有与糖基转移酶A亚单位特异性结合的活性，并中和糖基转移酶A的活性，可以用于检测艰难梭菌，也可用用于制备治疗抗艰难梭菌的药物；
2、本发明提供的抗糖基转移酶A亚单位的纳米抗体还具有分子量小、亲和力高、结构和性能稳定等特点；能够耐受胃部酸性环境，不易被胃蛋白酶降解等优势；
3、本发明提供的抗糖基转移酶A亚单位的纳米抗体制备成本低廉，能大大降低抗体生产成本。
附图说明
图1为抗糖基转移酶A亚单位的纳米抗体的筛选结果示意图；
图2为抗糖基转移酶A亚单位的纳米抗体纯化后的SDS-PAGE分析结果示意图；
图3为纳米抗体特异性的Elisa检测结果示意图。
具体实施方式
以将结合附图对本发明各实施例的技术方案进行清楚、完整的描述，显然，所描述实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例，都属于本发明。
实施例
羊驼血清中存在一种天然缺失轻链的抗体，即重链抗体(heavy-chain antibody,hcAb)。单域重链抗体(single domain antibody,sdAb)是指仅由重链抗体可变区(Variable region)组成的基因工程抗体，又称为VHH抗体(variable domain of heavychain of heavy-chain antibody,VHH antibody)或纳米抗体(Nanobody,Nb)。与传统抗体相比，单域重链抗体具有分子量小，抗逆性强，在强酸条件下仍具有高活性，因此这非常适合通过胃投递进入肠道，同时保持了良好的活性参与抑制艰难梭菌。本发明利用噬菌体展示技术，从羊驼噬菌体文库中筛选出能够与靶分子糖基转移酶A亚单位结合的噬菌体阳性克隆，从而获得了抗糖基转移酶A亚单位的纳米抗体核酸序列，并且能够在大肠杆菌中高效表达，从而纯化获得了目的抗体，即抗糖基转移酶A亚单位的纳米抗体。
下面结合具体实施例，对具体操作进行详细阐述。
(一)抗糖基转移酶A亚单位的纳米抗体的筛选
(1)将具有RBD氨基酸序列的糖基转移酶A亚单位蛋白用10mM PBS稀释至终浓度10μg/mL，按100μl每孔加入酶标板中，于37℃包被2h；该糖基转移酶A亚单位蛋白的制备可以参见Clostridium difficile chimeric toxin receptor binding domain vaccineinduced protection against different strains in active and passive challengemodels，Jing-Hui Tian et al.Vaccine 35(2017)4079-4087。
(2)弃掉包被液，每孔加入300μL 10mM PBS洗涤3次；再每孔加入300μL含5％脱脂奶粉的10mM PBS封闭1h；
(3)弃掉封闭液，每孔加入300μL10mM PBST(其中含体积浓度为0.1％的Tween)洗涤3次，每孔再加入300μL10mM PBS洗涤3次；
(4)加入50μL羊驼噬菌体文库(最初使用的羊驼噬菌体文库购自金开瑞生物工程有限公司)和50μL含5％脱脂奶粉的10mM PBS，于37℃孵育2h；
(5)弃掉上清，用300μL PBST洗涤6次，每次间隔1min，然后再用300μL 10mM PBS洗涤3次；
(6)弃尽上清，加入pH＝2.2的Gly-HCl洗脱液静置洗脱8min；再加入pH＝9.1Tris-HCl缓冲液中和；当所得洗脱物在OD600nm下吸光值约为0.6时，将其感染大肠杆菌TG1(Lucigen，60502-1)，并于37℃、220rpm转速下感染1h，之后取10μL感染后的菌液，利用2YT培养基(含1.6％(W/V)Tryptone、1％(W/V)Yeast Extract、0.5％(W/V)NaCl)进行10倍梯度稀释，然后每个梯度取10μL通过流线测滴度，并将剩余菌液全部涂至大的氨苄平板上，于37℃培养过夜；
(7)用2YT培养基刮取过夜的菌体收集至EP管中，用于下一轮淘选用的羊驼噬菌体文库。
上述步骤(1)-(7)为第一轮淘选，接下来按照相同的步骤进行第二轮淘选和第三轮淘选；不同之处在于，在第二轮淘选过程中，所涉及的每处10mM PBST洗涤遍数为12遍；在第三轮淘选过程中，所涉及的每处10mM PBST洗涤遍数为18遍。
三轮淘选结果见图1所示，随着筛选轮数的增加，滴度逐渐升高，说明筛选过程中，有序列富集，筛选过程正常。
(二)特异性噬菌体阳性克隆的鉴定
(1)第三轮筛选结束后，将第三轮步骤(6)中所得洗脱物(OD600nm下吸光值约为0.6)感染大肠杆菌TG1(购自Lucigen，60502-1)，于37℃、220rpm转速下感染1h，之后取10μL菌液10倍稀释后涂于平板上，再于37℃倒置培养过夜；
(2)按照2YT、0.2％葡萄糖、100μg/ml氨苄配制2YT+葡萄糖+氨苄培养基，然后平铺于96孔板中；用枪头挑取(1)中所述平板中的单克隆置于平铺有培养基的96孔板中，于37℃、220rpm转速下培养4h；然后取100μL上述96孔板中的菌液置于新的96孔板中，并在其中加入50μL辅助噬菌体M13K07(购自Biolabs，N03155)，于37℃、220rpm转速下培养1h；
(3)将培养所得菌液于4000rpm离心8min后弃掉上清，换成A+K培养基(为含1.6％(W/V)Tryptone，1％(W/V)Yeast Extract，0.5％(W/V)NaCl，100μg/ml Amp，50μg/mlKana)，于37℃、220rpm转速下培养过夜；
(4)将培养所得菌液于4000rmp离心8min，取上清加入到包被有TcdA蛋白的酶标板(见(一)中有关糖基转移酶A亚单位蛋白包被操作)中，于37℃孵育1h；
(5)按照每孔400μL，先用10mM PBST洗涤3遍，再用10mM PBS洗涤3遍，每次洗涤完后均需要拍干；
(6)按照每孔100μL，加入含5％脱脂奶粉的10mM PBS稀释的辣根过氧化物酶标记的抗M13抗体(购自义翘神州公司)，于37℃孵育40min；
(7)按照每孔400μL，先用10mM PBST洗涤3遍再用10mM PBS洗涤3遍，每次洗涤完后均需要拍干；
(8)按照每孔100μL，加入TMB显色液(购自Solarbio，PR1210)，于37℃避光反应15min；之后每孔50μL，加入2M H2SO4终止液，终止反应，并于450nm下测定各孔吸光度。
分别对第二轮淘选所得噬菌体(即第二轮步骤(6)中所得洗脱物(OD600nm下吸光值约为0.6))和第三轮挑选所得噬菌体按照步骤(1)-(3)进行富集，按照步骤(4)-(8)经ELISA效价检测(即于450nm下对各板孔测量吸收值)，测量结果见表1所示。
从表1可以看出，第三轮富集后，阳性率和结合效价均有较高提升。
表1特异性噬菌体阳性克隆鉴定表
挑取阳性克隆测序分析，可以确定编码为SEQ ID NO.7、SEQ ID NO.8和SEQ IDNO.9所示；可变区氨基酸序列为SEQ NO.4-SEQ NO.6所示；其中，SEQ NO.4第101-115位氨基酸序列为SEQ NO.1所示的CDR-1，SEQ NO.5第101-115位氨基酸序列为SEQ NO.2所示的CDR-2，SEQ NO.6第101-115位氨基酸序列为SEQ NO.3所示的CDR-3。
(三)抗糖基转移酶A亚单位的纳米抗体的表达
(1)采用NcoI/Not I对得到的核苷酸序列SEQ NO.7-SEQ NO.9进行双酶切，与经过相同酶切的pEt 25b(+)载体连接，将连接好的质粒电转化至大肠杆菌Rosetta DE3表达菌株中，并挑取单克隆菌株接种于4mL LB-Amp培养基(为含1％Tryptone，0.5％YeastExtract，1％NaCl，100μg/ml Amp的去离子水)，于37℃、220rpm转速下培养5h；
(2)按照1％(V/V)接种于100mL LB-Amp-Glu培养基(含1％Tryptone，0.5％YeastExtract，1％NaCl，0.2％Glu，100μg/ml Amp)，于37℃、220rpm转速下培养至在OD600nm下吸光值约为约0.5；之后，加入终浓度为0.1mM的IPTG(购自Amresco公司)，于30℃、220rpm转速下诱导过夜；
(3)将诱导所得产物于12000rpm离心10min，之后弃上清，收集菌体；
(4)每1g菌液用30mLBuffer A计量，向收集菌体中加入Buffer A(50mM PB，300mMNaCl)重悬菌体；
(5)对重悬后的菌体进行超声破碎；超声条件为：25～35min、超声5s间隔7s、功率35％；
(6)超声破碎后的菌液于4℃、12000rpm转速下离心20min，取上清，过0.45μm滤膜，收集菌液用于抗糖基转移酶A亚单位的纳米抗体纯化。
(四)抗糖基转移酶A亚单位的纳米抗体的纯化
采用层析柱对超声破碎、过滤后的菌液进行纯化，具体操作为：
(1)清洗：开机，清洗各层析柱通路，设定柱压限为0.45MPa；
(2)平衡：按照8mL/min，以一级水冲洗，清洗填料，至基线平稳；然后，按照8mL/min换Buffer B(50mM PB，300mM NaCl，500mM咪唑)液冲洗至基线平稳；再按照8mL/min，换Buffer A(50mM PB，300mM NaCl)液冲洗至基线平稳。
(3)上样：按照8mL/min，将菌液加入层析柱；
(4)复平衡：上样完成后，按照8mL/min，以Buffer A液复平衡层析柱，冲洗至基线完全平稳；
(5)洗脱：按照8mL/min，以90％Buffer A+10％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
进一步，按照8mL/min，使用80％Buffer A+20％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
进一步，按照8mL/min，使用70％Buffer A+30％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
进一步，按照8mL/min，使用60％Buffer A+40％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
进一步，按照8mL/min，使用50％Buffer A+50％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
进一步，按照8mL/min，使用100％Buffer B冲洗层析柱，收集280nm吸收值高于100mAU的洗脱液；
完成对表达后的抗糖基转移酶A亚单位的纳米抗体的纯化；
(6)清洗：使用100％Buffer B冲洗层析柱至基线平稳，更换一级水冲洗层析柱至基线平稳；再使用含20％乙醇的水溶液冲洗层析柱至基线平稳，取下层析柱，并于4℃保存。
对纯化后的抗糖基转移酶A亚单位的纳米抗体进行SDS-PAGE分析，如图2所示，1-3泳道为纯化后纳米抗体。结果表明抗糖基转移酶A亚单位的纳米抗体相对分子质量约为15KD，与理论分子质量相符，且纯化后的条带无明显杂带，表明纳米抗体纯度较高，
(五)Elisa检测上述纳米抗体的特异性
(1)取纯化后的抗糖基转移酶A亚单位的纳米抗体样品，按100μl/孔，分别向糖基转移酶A亚单位(TcdA蛋白)包被的酶标板(见(一)中有关糖基转移酶A亚单位蛋白包被操作)、BSA包被的酶标板(与TcdA蛋白包被的酶标板制作方法相同)及卵清蛋白包被的酶标板(与TcdA蛋白包被的酶标板制作方法相同)加入样品，于37℃放置60min；
(2)按照300ul/孔，先用PBST清洗3次；按照300ul/孔，再用PBS洗3次，最后一次洗完后彻底拍干板内液体；
(3)按100ul/孔，向酶标板加入抗体(抗体为以1:5000利用抗体稀释液(10mM PBS，5％脱脂奶粉)稀释后的二抗)，于37℃放置60min；
(4)按照300ul/孔，先用PBST清洗3次；按照300ul/孔，再用PBS洗3次，最后一次洗完后彻底拍干板内液体；
(5)按100μl/孔，加入TMB显色液(购自Solarbio，PR1210)，于37℃避光显色15min；之后按50μl/孔，加入2M H2SO4终止反应，并于450nm测定各孔吸光度，结果见图3所示。
从图中可以看出，分别使用三种纳米抗体对糖基转移酶A亚单位、BSA、卵清蛋白(即OVA)三种抗原包被板进行检测，本实施例得到的纳米抗体能够特异性的结合糖基转移酶A亚单位。
本领域的普通技术人员将会意识到，这里所述的实施例是为了帮助读者理解本发明的原理，应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合，这些变形和组合仍然在本发明的保护范围内。
序列表
<110> 四川大学华西医院
<120> 一种抗糖基转移酶A亚单位的纳米抗体及其应用
<130> 202101
<150> 2020113828788
<151> 2020-11-30
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caggctccag ggaaggagcg tgagttcgta gcagcgattt cttcttctga cgactctact 180
aactatgcag actccgtgaa gggccgattc accatctcca gagacaacgc ccagaacacg 240
gtgtatcttc aaatgaacag cctgaaacct gaggacacgg ccgtttatta ctgtgcagcc 300
cactactacc gttacgacgg ttctggcctg tacctgtacg actactgggg tgagggtact 360
caggtcaccg cctcctcagc ggccgcatac ccgtacgacg ttgcggacta cggttcccac 420
catcatcatc atcactatac tgttgaaagt tga 453
<210> 9
<211> 438
<212> DNA
<213> Lama pacos
<400> 9
atggcccagg tgcagctgca ggagtctgga ggaggcttgg tgcaggctgg agggtctctg 60
agactctctt gtgccgccag cggatctact ttcaactcta acgctatggg ctggttccgc 120
caggctccag ggaaggagcg tgagttcgta gcagcgattt cttctgacgg ttcttctact 180
aactatgcag actccgtgaa gggccgattc accatctcca gagacaacgc caagaacacg 240
gtgtatcttc aaatgaacag cctgaaacct gaggacacgg ccgtttatta ctgtgcagcc 300
aggcggccgt ttattactgg ttctacacgg agaccatctg accggtgggg tcagggtact 360
caggtcaccg tctcctcagc ggccgcatac ccgtacgacg ttccggacta cggttcccac 420
caccatcacc atcactag 438
Claims (4)
1.一种抗糖基转移酶A亚单位的纳米抗体，其特征在于，其具有SEQ ID NO.6所示可变区中的互补决定区CDR-1、CDR-2、CDR-3。
2.根据权利要求1所述抗糖基转移酶A亚单位的纳米抗体，其特征在于，所述纳米抗体的可变区序列由SEQ ID NO.6所示的氨基酸序列组成。
3.一种编码权利要求2所述的纳米抗体的核苷酸，其特征在于，所述核苷酸的序列由SEQ ID NO.9所示。
4.权利要求2所述抗糖基转移酶A亚单位的纳米抗体在制备艰难梭菌检测试剂中的应用。
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