CN113698477B - 一种抗SARS-CoV-2单链抗体及其制备方法和应用 - Google Patents
一种抗SARS-CoV-2单链抗体及其制备方法和应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种抗SARS‑CoV‑2单链抗体及其制备方法和应用。所述抗SARS‑CoV‑2单链抗体的互补决定区包括CDR1和CDR3,所述CDR1包括SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列,所述CDR3包括SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。所述抗SARS‑CoV‑2单链抗体具备高亲和力,高效和SARS‑CoV‑2结合,不易解离,具备高病毒中和活性,且可以在原核系统中高效表达,表达流程标准可控,可大大降低生产成本,同时,分子量小,理化性质稳定,耐热性好,可大大降低作为防治药物的运输成本,在防治SARS‑CoV‑2领域具有重要应用价值。
Description
技术领域
本发明属于生物医药技术领域,涉及一种抗SARS-CoV-2单链抗体及其制备方法和应用。
背景技术
新型冠状病毒SARS-CoV-2是由世界卫生组织命名的一种新型的β属的冠状病毒,该病毒有包膜,颗粒呈圆形或椭圆形,主要结构蛋白包括刺突蛋白(Spike,S)、信使蛋白(Envelop,E)、膜蛋白(Membrane,M)和核衣壳蛋白(Nucleocapsid,N),有研究发现SARS-CoV-2通过与血管紧张素转换酶2(Angiotensin converting enzyme 2,ACE2)受体结合进入宿主细胞,进行病毒的感染和复制,引发肺损伤,肺炎,严重可导致严重急性呼吸系统综合征,脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍及多器官功能衰竭等。
目前主要依赖于疫苗的预防作用防治SARS-CoV-2,但SARS-CoV-2能够发生突变产生新的致病突变体,使得疫苗的保护效果降低,因此需要针对COVID-19快速有效的预防和治疗策略。
除了疫苗,许多研究证明中和性抗体在机体抵抗病毒感染中起到关键性作用,含有中和抗体的恢复期血清已被用于治疗COVID-19患者。
CN112442120A公开了一种抗严重急性呼吸系统综合征II型冠状病毒SARS-CoV-2的中和抗体,从COVID-19康复患者血液中提取外周免疫细胞,并从中筛选可以和新冠病毒抗原蛋白-刺突蛋白结合的B细胞,然后对已生产抗体的单个B细胞在单细胞水平上进行分析,获得B细胞内编码中和抗体可变区重链和轻链的基因序列,利用所述序列重新构建和表达中和抗体,有望用于新冠病毒所引起的肺炎等疾病的治疗和预防,但由于缺乏来源和潜在的副作用而受到限制,并且人源中和抗体生产成本高,运输条件严格,免疫剂量大以及可能的ADE副作用,难以大规模应用。
纳米抗体是一种在骆驼和鲨鱼中发现的单链抗体,近年来受到了广泛的关注,纳米抗体分子量小、亲和力高、热稳定性强且生产成本低,有望成为未来治疗性抗体发展的重要方向。
综上所述,提供一种亲和力高、热稳定性强且生产成本低的抗SARS-CoV-2抗体,对于SARS-CoV-2防治领域具有重要意义。
发明内容
针对现有技术的不足和实际需求,本发明提供一种抗SARS-CoV-2单链抗体及其制备方法和应用,所述抗SARS-CoV-2单链抗体具备高亲和力,且可以在原核系统中高效表达,生产成低本,理化性质稳定,耐热性好,运输成本低,在防治SARS-CoV-2领域具有重要应用价值。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种抗SARS-CoV-2单链抗体,所述抗SARS-CoV-2单链抗体的互补决定区包括CDR1和CDR3,所述CDR1包括SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列,所述CDR3包括SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。
本发明的抗SARS-CoV-2单链抗体具备高亲和力,高效和SARS-CoV-2结合,不易解离,且可以在原核系统中高效表达,表达流程标准可控,可大大降低生产成本,同时,分子量小,理化性质稳定,耐热性好,可大大降低作为防治药物的运输成本,在防治SARS-CoV-2领域具有重要应用价值。
根据本发明,所述抗SARS-CoV-2单链抗体还包括高变区HV2和HV4,所述HV2包括SEQ ID NO.5或SEQ ID NO.6所示的氨基酸序列,所述HV4包括SEQ ID NO.7所示的氨基酸序列。
优选地,所述抗SARS-CoV-2单链抗体还包括骨架区FR1、FR2和FR3。
优选地,所述FR1包括SEQ ID NO.8或SEQ ID NO.9所示的氨基酸序列。
优选地,所述FR2包括SEQ ID NO.10或SEQ ID NO.11所示的氨基酸序列。
优选地,所述FR3包括SEQ ID NO.12或SEQ ID NO.13所示的氨基酸序列。
优选地,所述抗SARS-CoV-2单链抗体包括SEQ ID NO.14或SEQ ID NO.15所示的氨基酸序列。
SEQ ID NO.1:DSPCSLDS。
SEQ ID NO.2:DSSCALDS。
SEQ ID NO.3:RAYSTTGDERDCRWQGYI。
SEQ ID NO.4:RAYSLSAGMCAWMGYI。
SEQ ID NO.5:ATKKENLS。
SEQ ID NO.6:ATKKESLS。
SEQ ID NO.7:NKASK。
SEQ ID NO.8:ERVEQTPTTTTKEAGESLTINCVLR。
SEQ ID NO.9:ERLEQTPTTTTKETGESLTINCVLR。
SEQ ID NO.10:TFWYFTKKGATKKE。
SEQ ID NO.11:TYWYFTKKGATKKE。
SEQ ID NO.12:NLSNGGRYAETVNKASKSFSLQISDLRVEDSGTYHC。
SEQ ID NO.13:SLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHC。
SEQ ID NO.14:
MAERVEQTPTTTTKEAGESLTINCVLRDSPCSLDSTFWYFTKKGATKKENLSNGGRYAETVNKASKSFSLQISDLRVEDSGTYHCRAYSTTGDERDCRWQGYIEGYGTILTVN。
SEQ ID NO.15:
MAERLEQTPTTTTKETGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCRAYSLSAGMCAWMGYIEGGGTTLTVN。
第二方面,本发明提供一种核酸分子,所述核酸分子包括编码第一方面所述抗SARS-CoV-2单链抗体的核酸序列。
优选地,所述核酸分子包括SEQ ID NO.16或SEQ ID NO.17所示的核酸序列。
SEQ ID NO.16:
atggccgaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattctccctgttcattggatagcacgttctggtatttcacaaaaaagggtgcaacaaagaaggagaacttatcaaatggcggacgatatgcggaaacagtgaacaaggcatcaaagtccttttctttacaaattagtgacctaagagttgaagacagtggtacatatcactgtagagcgtatagcaccaccggggatgagagggactgtaggtggcagggctatattgaaggatacggcaccattctgactgtgaat。
SEQ ID NO.17:
atggccgaacggcttgaacaaacaccgacaacgacaacaaaggagacaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtagagcgtatagcctttcagctgggatgtgtgcctggatgggctacattgaaggaggcggcaccactctgactgtgaat。
第三方面,本发明提供一种重组载体,所述重组载体包括第二方面所述的核酸分子。
第四方面,本发明提供一种重组细胞,所述重组细胞包括第二方面所述的核酸分子或第三方面所述的重组载体。
第五方面,本发明提供一种第一方面所述的抗SARS-CoV-2单链抗体的制备方法,所述制备方法包括以下步骤:
(1)使用SARS-CoV-2S1蛋白免疫条纹斑竹鲨,分离免疫后条纹斑竹鲨的外周血单核细胞并提取总RNA,以所述RNA为模板逆转录制备cDNA;
(2)以所述cDNA为模板,PCR扩增单链抗体可变区,构建噬菌体展示文库;
(3)在所述噬菌体展示文库中筛选具有SARS-CoV-2抗原特异性的噬菌体抗体,并进行测序,获得抗体的核酸序列;
(4)利用所述抗体的核酸序列构建表达载体,并转入细胞进行表达及纯化,得到所述抗SARS-CoV-2单链抗体。
优选地,步骤(3)所述抗体的核酸序列为如SEQ ID NO.16或SEQ ID NO.17所示的核酸序列。
优选地,步骤(4)所述构建表达载体包括构建原核表达载体或真核表达载体。
优选地,所述原核表达载体转化入大肠杆菌进行表达。
优选地,所述真核表达载体转入293细胞进行表达。
第六方面,本发明提供一种药物组合物,所述药物组合物包括第一方面所述的抗SARS-CoV-2单链抗体、第二方面所述的核酸分子、第三方面所述的重组载体或第四方面所述的重组细胞中的任意一种或至少两种的组合。
优选地,所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
第七方面,本发明提供如第一方面所述的抗SARS-CoV-2单链抗体、第二方面所述的核酸分子、第三方面所述的重组载体、第四方面所述的重组细胞或第六方面所述的药物组合物在制备抗SARS-CoV-2药物中的应用。
与现有技术相比,本发明具有以下有益效果:
(1)本发明的抗SARS-CoV-2单链抗体具备高亲和力,高效和SARS-CoV-2结合,不易解离,且制造成本和运输成本均较低,在防治SARS-CoV-2领域具有重要应用价值;
(2)本发明的抗SARS-CoV-2单链抗体能够以高亲和力和SARS-CoV-2病毒RBD蛋白结合,且具备高病毒中和活性。
附图说明
图1A为SARS-CoV-2单链抗体20G6纯化后蛋白电泳图,左1泳道为Maker,之后泳道为多次技术重复;
图1B为SARS-CoV-2单链抗体17F6纯化后蛋白电泳图,左1泳道为Maker,之后泳道为多次技术重复;
图2A为ELISA检测20G6抗体与不同突变SARS-CoV-2病毒RBD蛋白结合活性图;
图2B为ELISA检测17F6抗体与不同突变SARS-CoV-2病毒RBD蛋白结合活性图;
图3A为20G6抗体与WH-Hu-1株病毒RBD蛋白亲和力图;
图3B为17F6抗体与WH-Hu-1株病毒RBD蛋白亲和力图;
图3C为20G6 Fc抗体与WH-Hu-1株病毒RBD蛋白亲和力图;
图3D为20G6 Fc抗体与Alpha株病毒RBD蛋白亲和力图;
图3E为20G6 Fc抗体与Beta株病毒RBD蛋白亲和力图;
图3F为20G6 Fc抗体与Kappa株病毒RBD蛋白亲和力图;
图3G为20G6 Fc抗体与Delta株病毒RBD蛋白亲和力图;
图3H为17F6 Fc抗体与WH-Hu-1株病毒RBD蛋白亲和力图;
图3I为17F6 Fc抗体与Alpha株病毒RBD蛋白亲和力图;
图3J为17F6 Fc抗体与Beta株病毒RBD蛋白亲和力图;
图3K为17F6 Fc抗体与Kappa株病毒RBD蛋白亲和力图;
图3L为17F6 Fc抗体与Delta株病毒RBD蛋白亲和力图;
图4为20G6、17F6、20G6 Fc和17F6 Fc抗体对WH-Hu-1株病毒中和抑制率图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例构建单链抗体噬菌体库,所述单链抗体噬菌体库的构建方法包括以下步骤:
(1)取100μg SARS-CoV-2S1蛋白(购自北京义翘神州)溶于250μL PBS中,并与等体积铝佐剂充分振荡乳化,皮下及肌肉多点注射免疫条纹斑竹鲨,共免疫6次,每次间隔2周;
(2)从第二次免疫开始,每次免疫一周后尾静脉采血1mL,将其中0.5mL经抗凝处理分离淋巴细胞,trizol裂解,-80℃保存备用;另外取0.5mL室温静置1小时,离心分离血清检测血清效价,最后一次免疫后尾静脉采血检测血清假病毒中和效价;
(3)取第3、4、5、6次免疫后的淋巴细胞裂解液,使用氯仿抽提总RNA,采用bio-rad公司的逆转录试剂盒(bio-rad,Cat:1708891)合成cDNA;
(4)以所述cDNA为模板,利用特异性引物,上游引物:GCGAGGAGGAGGCCCAGCCGGCCATGGCCSMACGGSTTGAACAAACACC下游引物:ATAAGAATGCGGCCGCWTTCACAGTCASARKGGTSCC(参考文献,doi:10.1016/j.molimm.2006.07.299)扩增单链抗体可变区,PCR反应体系包括:
PCR扩增程序为:
98℃预变性30s;
98℃变性10s、61℃退火30s、72℃延伸30s,27个循环;
72℃延伸7min;
(5)使用1%琼脂糖凝胶电泳鉴定PCR产物,并进行回收纯化(Magen DNA凝胶回收试剂盒),得单链抗体可变区PCR产物,将单链抗体可变区PCR产物与pcantab5e噬菌粒载体分别进行NotI和SfiI双酶切,按载体150ng,片段50ng的比例于16℃连接过夜,连接产物经纯化后电转化入TG1感受态中,涂板,并同时做10倍倍比梯度稀释计算库容,37℃过夜培养,用涂布棒刮取平板上的菌落,重悬于培养基中,加入终浓度20%的甘油,-80℃保存备用,挑取96个克隆进行菌液PCR鉴定文库阳性转化率并测序验证抗体多样性,经鉴定,库容达2×108,阳性转化率100%,文库多样性大于90%。
实施例2
本实施例进行SARS-CoV-2单链抗体筛选及表达,包括以下步骤:
(1)取100μL实施例1制备的单链抗体噬菌体库,接种于50mL含有氨苄抗生素和1%葡萄糖的2YT培养基中,37℃培养至对数期,加入20倍菌数的辅助噬菌体M13KO7,混匀后37℃静置20分钟,再振荡培养30分钟,离心,弃掉培养基,加入50mL 2YT氨苄霉素卡那霉素培养基,30℃振荡培养过夜,第二天离心,收集上清,加入1/4体积的PEG/NaCl,4℃沉淀重组噬菌体2小时,离心收集噬菌体沉淀,使用5mL PBS溶解,重复沉淀1次,PBS溶解噬菌体,加入终浓度为15%的甘油,分装保存于-80℃备用,同时取10μL做10倍梯度稀释,感染对数期TG1,培养过夜,计算滴度;
(2)RBD抗原用CBS缓冲液稀释至10μg/mL,充分混匀后加入96孔酶标板中,100μL/孔,4℃孵育12小时,弃掉抗原,加入封闭液(5%脱脂牛奶溶解于PBST中),200μL/孔,37℃封闭2小时,洗板,0.05%PBST,200μL/孔,洗4次,2分钟/次,甩掉液体,拍干,备用;
(3)第一轮筛选,取100倍库容的重组噬菌体稀释于100μL封闭液中,加入包被了RBD的酶标孔中,室温孵育2小时,弃掉噬菌体,使用0.1%PBST洗涤10次,2分钟/次,100μL三乙胺洗脱10分钟,取出洗脱液,立即加入1M Tris-HCl(pH 7.4)中和洗脱液,中和后的洗脱液加入3mL对数生长期TG1中,静置30分钟,振荡培养30分钟,取10μL做10倍梯度稀释,计算库容,剩下全部离心,留300μL培养基将菌体重悬,涂于2YT固体培养基平板,37℃倒置培养过夜,第二天,梯度稀释的平板计算库容,菌库平板用涂布棒将菌体刮下,重悬于3mL 2YT培养基中,加入终浓度为20%的甘油,-80冻存备用,得到第一轮菌种库,取100μL第一轮菌种库于50mL培养基中,37℃振荡培养至对数期,并按步骤(1)所述制备第一轮重组噬菌体库;
(4)第二轮筛选,具体步骤如步骤(3)中所述,在第一轮基础上改变条件,洗涤次数增加15次,包被抗原量降低3倍;
(5)第三轮筛选,具体步骤如步骤(3)中所述,在第二轮的基础上改变条件,洗涤次数增加15次,包被抗原量降低3倍,从第三轮筛选获得的库中挑选单个克隆,进行PhageELISA验证;
(6)Phage ELISA验证,挑取单个克隆于96孔板中,37℃振荡培养过夜,第二天取5μL菌液于新的96孔板中(300μL培养基/孔),37℃振荡培养至对数期,加入20倍菌量的辅助噬菌体,37℃静置30分钟,再振荡培养30分钟,离心,去掉培养基,加入300μL 2YT氨苄卡那培养基,30℃振荡培养过夜,取100μL上清于包被了RBD抗原(50ng/孔)的酶标板中,室温孵育2小时,同时用重组噬菌体库作为阳性对照,辅助噬菌体作为阴性对照,PBST洗板4次,加入HRP标记的抗M13抗体(购于成都阿帕克),100μL/孔,37℃孵育1小时,PBST洗板6次,加入TMB显色底物(购自millipore),100μL/孔,37℃避光孵育15分钟,加入50μL 1M H2SO4终止,检测450nm吸光值;
(7)原核表达:将Phage ELISA验证的阳性克隆进行测序,获得序列不同的抗体,PCR扩增出不同的抗体序列,上游带有NcoI酶切位点,下游带有XhoI酶切位点,回收PCR产物后,同时用NcoI和XhoI将PCR产物与PET28a载体分别进行双酶切,酶切体系为:1μg PCR产物和1μg质粒,无菌水补至50μL,37℃酶切20小时,胶回收,取150ng载体与50ng片段,16℃连接3小时,连接产物加入大肠杆菌TOP 10感受态中,冰置30分钟,42℃热激90s,立即冰置3分钟,加入500μL LB培养基复苏60分钟,涂板,37℃静置培养过夜,第二天每个平板挑取5个克隆进行菌落PCR,并测序鉴定抗体片段是否正确插入,抗体片段插入正确的克隆接入10mLLB卡那培养基中,振荡培养过夜,提取质粒,转化入大肠杆菌表达菌Arctic express中,挑取单个克隆,于LB培养基中培养至对数期,加入0.1mM IPTG,16℃诱导表达20小时,收集菌体沉淀,高压破碎,离心,收集上清,进行Ni柱亲和纯化,得到的粗提物再进行离子交换和分子筛,最终获得SARS-CoV-2单链抗体20G6(SEQ ID NO.14)和17F6(SEQ ID NO.15),纯度>95%,纯化后蛋白电泳图如图1A和1B所示;
真核表达:以pET28a-20G6和pET28a-17F6为模板扩增20G6与17F6,上游带有AfeI酶切位点,下游带有NheI酶切位点,胶回收PCR产物后,同时用AfeI和NheI将PCR产物与PCMV-hIgG1Fc载体分别进行双酶切,酶切体系为:1μg PCR产物和1μg质粒,无菌水补至50μL,37℃酶切20小时,胶回收,取150ng载体与50ng片段,16℃连接3小时,胶回收,取150ng载体与50ng片段,16℃连接3小时,连接产物加入大肠杆菌TOP 10感受态中,冰置30分钟,42℃热激90s,立即冰置3分钟,加入500μL LB培养基复苏45分钟,涂板,37℃静置培养过夜,第二天每个平板挑取5个克隆进行菌落PCR,并测序鉴定抗体片段是否正确插入,抗体片段插入正确的克隆接入200mL LB氨苄培养基中,振荡培养过夜,去内毒素提取质粒,将400μg质粒与800μg PEI转染试剂分别重悬于5mL Opti-MEM培养基中,混合,静置20min,加入200mL对数生长期的expi293细胞中,37℃、5%CO2培养5天,离心收集培养上清,Protein A亲和层析,获得Fc标签融合表达的20G6 Fc抗体和17F6 Fc抗体,抗体纯度>95%。
实施例3
本实施例检测实施例2制备的SARS-CoV-2单链抗体结合能力,包括以下步骤:
(1)分别将五种不同毒株RBD(刺突蛋白中受体结合位点结构域,为第332~527位氨基酸)蛋白(WH-Hu-1株,英国株B.1.1.7(Alpha,包含N501Y突变位点),南非株B.1.351(Bbeta,包含K417N、E484K、N501Y三个位点突变)和印度株B.1.617(Kappa,包含L452R,E484Q突变位点)、B.1.617.2(Delta,包含L452R,T478K突变位点))稀释于CBS缓冲液中,包被酶标板,50ng/100μL/孔,4℃孵育12小时,弃掉抗原,加入封闭液(5%脱脂牛奶溶解于PBST中),200μL/孔,37℃封闭2小时,洗板,0.05%PBST,200μL/孔,洗4次,2分钟/次,甩掉液体,拍干;
(2)分别将20G6与17F6抗体稀释于PBST中,从100μg/mL开始,做半对数稀释,共稀释12个梯度,100μL/孔加入酶标板中,37℃孵育2小时,PBST洗板4次,2分钟/次,加入HRP标记的抗人IgG二抗,37℃孵育1小时,PBST洗板6次,2分钟/次,甩掉液体,拍干,加入TMB显色底物,100μL/孔,37℃避光孵育15分钟,加入50μL 1M H2SO4终止,检测450nm吸光值,计算抗原抗体结合EC50,结果如图2A和图2B所示,20G6抗体的EC50为0.003μg/mL,17F6 EC50为0.007μg/mL。
实施例4
本实施例检测实施例2制备的SARS-CoV-2单链抗体亲和力。
利用生物膜干涉技术(BLI)检测单链抗体亲和力,使用gator bio BLI设备,分别将20G6、17F6、20G6Fc和17F6 Fc单链抗体按10μg/mL稀释于PBS缓冲液中(含0.02%tween-20和0.2%BSA),五种RBD蛋白(WH-Hu-1,Alpha,Beta,Kappa,Delta)的浓度梯度为200nM、100nM、50nM、25nM、12.5nM、6.25nM和3.125nM,结果如图3A-图3L所示,根据结合曲线计算结合常数Ka,解离常数Kd,和亲和力常数KD,如表1所示。
表1
由表1可知,本发明通过原核表达和真核表达制备的SARS-CoV-2单链抗体均具备高亲和力。
实施例5
本实施例检测实施例2制备的SARS-CoV-2单链抗体体外病毒中和活性。
分别将20G6及17F6单链抗体进行梯度稀释,从10μg/mL开始做4倍梯度稀释,共稀释5个梯度,加入96孔板中,60μL/孔,分别与100TCID50的WH-Hu-1株SARS-CoV-2病毒或南非株SARS-CoV-2病毒(Beta)混合,37℃孵育2小时,取100μL抗体病毒混合物加入提前在96孔细胞板中培养的Vero E6细胞中,37℃5%CO2培养96小时,40倍镜下观察细胞病变效应(CPE)现象,以不出现CPE的最大稀释倍数为终点计算IC100,结果如表2所示,所有体外病毒中和实验均在P3实验室中完成。
表2
由表2可知,CPE方法检测20G6抗体和17F6抗体完全中和100TCID50 WH-Hu-1株SARS-CoV-2(IC100)的浓度分别为156ng/mL和1.25ng/mL,完全中和100TCID50南非株SARS-CoV-2(IC100)的浓度分别为312ng/mL和1250ng/mL,表明本发明的单链抗体能有效结合并中和SARS-CoV-2突变株感染细胞。
实施例6
本实施例检测SARS-CoV-2单链抗体体外病毒中和活性(FRNT),包括以下步骤:
(1)第一天:Vero E6细胞细胞铺板,96孔板,2×104细胞/孔;
(2)第二天:将抗体稀释至浓度为100μg/mL,然后做4倍连续稀释,共9个梯度;
(3)将WH-Hu-1株SARS-CoV-2病毒用DMEM稀释至200FFU/25μL。
(4)取75μL病毒与75μL梯度稀释的抗体充分混匀,37℃、5%CO2孵育1小时;
(5)弃掉细胞培养液,取50μL病毒抗体混合液加入细胞板中,每个梯度3个复孔,轻微晃动,使液体均匀覆盖细胞,37℃、5%CO2孵育1小时;
(6)弃掉病毒抗体混合液,加入预热的1.6%CMC,100μL/孔,37℃、5%CO2孵育24小时;
(7)细胞固定、染色:
1)加入200μL/孔4%多聚甲醛,固定1小时;
2)弃掉培养基及固定液,再加满4%多聚甲醛,紫外照射30分钟;
3)弃固定液,用PBS洗板3次,200μL/孔;
4)用1%BSA含0.2%triton封闭,50μL/孔,室温放置30min,PBST洗3次;
5)一抗:1%BSA稀释兔抗SARS-N多克隆抗体,50μL/孔,37℃孵育1小时,PBST洗3次;
6)二抗:1%BSA稀释羊抗兔IgG-HRP,50μL/孔,37℃孵育1小时,PBST洗3次;
7)显色:TrueBlue 50μL/well,室温放置10min,ddH2O洗板三次,甩干,计数;
(8)计算中和抑制率:抑制率=[1-实验孔病灶数目/病毒对照组病灶数目]×100%,Graphpad软件拟合抗体中和曲线,如图4所示,并计算IC50,结果如表3和图4所示。
表3
IC50(ng/mL) | |
20G6 | 370(24.67nM) |
17F6 | 2562(170.8nM) |
20G6 Fc | 860(8.6nM) |
17F6 Fc | 3647(36.5nM) |
由表3可知,FRNT检测20G6和17F6对200FFU WH-Hu-1株SARS-CoV-2病毒体外中和IC50分别为370ng/mL和2562ng/mL,20G6 Fc和17F6 Fc对200FFU WH-Hu-1株SARS-CoV-2病毒体外中和IC50分别为860ng/mL和3647ng/mL,表明本发明的单链抗体能有效结合并中和SARS-CoV-2突变株感染细胞。
综上所述,本发明的抗SARS-CoV-2单链抗体能够以高亲和力结合SARS-CoV-2病毒RBD蛋白,且几乎不解离,能有效结合并中和SARS-CoV-2突变株感染细胞,且制造成本和运输成本均较低,在防治SARS-CoV-2领域具有重要应用价值。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 厦门福宸百奥生物技术有限公司,厦门联合呼吸健康研究院,广州医科大学
<120> 一种抗SARS-COV-2单链抗体及其制备方法和应用
<130> 20210726
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Claims (14)
1.一种抗SARS-CoV-2纳米抗体,其特征在于,所述抗SARS-CoV-2纳米抗体的互补决定区由CDR1和CDR3组成;
所述CDR1为SEQ ID NO.1所示的氨基酸序列,所述CDR3为SEQ ID NO.3所示的氨基酸序列;
或,所述CDR1为SEQ ID NO.2所示的氨基酸序列,所述CDR3为SEQ ID NO.4所示的氨基酸序列。
2.根据权利要求1所述的抗SARS-CoV-2纳米抗体,其特征在于,所述抗SARS-CoV-2纳米抗体还包括高变区HV2和HV4;
所述HV2为SEQ ID NO.5或SEQ ID NO.6所示的氨基酸序列;
所述HV4为SEQ ID NO.7所示的氨基酸序列。
3.根据权利要求1所述的抗SARS-CoV-2纳米抗体,其特征在于,所述抗SARS-CoV-2纳米抗体还包括骨架区FR1、FR2和FR3;
所述FR1为SEQ ID NO.8或SEQ ID NO.9所示的氨基酸序列;
所述FR2为SEQ ID NO.10或SEQ ID NO.11所示的氨基酸序列;
所述FR3为SEQ ID NO.12或SEQ ID NO.13所示的氨基酸序列。
4.根据权利要求1所述的抗SARS-CoV-2纳米抗体,其特征在于,所述抗SARS-CoV-2纳米抗体为SEQ ID NO.14或SEQ ID NO.15所示的氨基酸序列。
5.一种核酸分子,其特征在于,所述核酸分子包括编码权利要求1-4任一项所述抗SARS-CoV-2纳米抗体的核酸序列。
6.一种重组载体,其特征在于,所述重组载体包括权利要求5所述的核酸分子。
7.一种重组细胞,其特征在于,所述重组细胞包括权利要求5所述的核酸分子或权利要求6所述的重组载体。
8.一种权利要求1-4任一项所述的抗SARS-CoV-2纳米抗体的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)使用SARS-CoV-2 S1蛋白免疫条纹斑竹鲨,分离免疫后条纹斑竹鲨的外周血单核细胞并提取总RNA,以所述RNA为模板逆转录制备cDNA;
(2)以所述cDNA为模板,PCR扩增纳米抗体可变区,构建噬菌体展示文库;
(3)在所述噬菌体展示文库中筛选具有SARS-CoV-2抗原特异性的噬菌体抗体,并进行测序,获得抗体的核酸序列;
(4)利用所述抗体的核酸序列构建表达载体,并转入细胞进行表达及纯化,得到所述抗SARS-CoV-2纳米抗体。
9.根据权利要求8所述的方法,其特征在于,步骤(4)所述构建表达载体包括构建原核表达载体或真核表达载体。
10.根据权利要求9所述的方法,其特征在于,所述原核表达载体转化入大肠杆菌进行表达。
11.根据权利要求9所述的方法,其特征在于,所述真核表达载体转入293细胞进行表达。
12.一种药物组合物,其特征在于,所述药物组合物包括权利要求1-4任一项所述的抗SARS-CoV-2纳米抗体、权利要求5所述的核酸分子、权利要求6所述的重组载体或权利要求7所述的重组细胞中的任意一种或至少两种的组合。
13.根据权利要求12所述的药物组合物,其特征在于,所述药物组合物还包括药学上可接受的载体。
14.如权利要求1-4任一项所述的抗SARS-CoV-2纳米抗体、权利要求5所述的核酸分子、权利要求6所述的重组载体、权利要求7所述的重组细胞或权利要求12所述的药物组合物在制备抗SARS-CoV-2药物中的应用。
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