CN116063469A - 一种寨卡病毒中和性纳米抗体及其制备方法与应用 - Google Patents
一种寨卡病毒中和性纳米抗体及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种寨卡病毒中和性纳米抗体及其制备方法与应用,本发明寨卡病毒中和性纳米抗体特异性地结合至寨卡病毒包膜蛋白的结构域III,特异性结合的抗原表位的氨基酸序列如SEQ ID NO:1所示;本发明筛选出的纳米抗体能够特异性识别寨卡病毒包膜蛋白结构域III上更为隐蔽的抗原位点,具有分子量小,仅由重链可变域组成,不含Fc段,但依然保持抗体序列的亲和力和特异性,对于寨卡病毒感染的预防或治疗具有重要意义。
Description
技术领域
本发明属于生物技术领域,具体涉及一种寨卡病毒中和性纳米抗体及其制备方法与应用。
背景技术
寨卡病毒(ZIKV)主要经蚊虫叮咬传播给人类,传播的媒介生物包括埃及伊蚊、白纹伊蚊等,其他非媒介传播途径包括母婴传播、性传播、器官移植传播、输血传播等。大部分的ZIKV感染者表现为无症状,其引起的急性症状潜伏期为3~14天,轻微的症状大约持续一周,包括发热、皮疹、关节痛、结膜炎等,这些症状在成人、儿童中表现相似。但在2013年的ZIKV大流行中观察到ZIKV感染率与吉兰-巴雷综合症(Guillain-Barre syndrome,GBS)发病率呈正相关,2015年的巴西ZIKV大流行发现妊娠期感染ZIKV会导致新生儿小头畸形发病率升高,由此,ZIKV感染受到人们的广泛关注。
ZIKV是一种单链RNA包膜病毒,全基因组长度为10,794kb,基因组编码7种非结构蛋白和3种结构蛋白,其中7种非结构蛋白为:NS1、NS2A、NS2B、NS3、NS4A、NS4B、NS5;3种结构蛋白为:衣壳(Capsid,C)蛋白、包膜(Envelope,E)蛋白、膜前体(Precursor of membrane,prM)蛋白。非结构蛋白主要参与病毒基因组的复制、翻译或调节宿主的免疫应答和代谢。NS1是病毒感染的主要抗原标志物,在病毒基因组复制和病毒粒子成熟过程中发挥重要作用。NS2A、NS2B、NS4A和NS4B是病毒组装的必需蛋白。NS3和NS5具备不同酶活性,与病毒基因组复制密切相关。结构蛋白主要参与病毒颗粒的组装、病毒吸附、诱导抗体产生保护性抗体等,位于病毒粒子表面的E蛋白是中和抗体识别的主要位点,它包含一个跨膜结构域和三个位于膜外的胞外结构域(EDI、EDII和EDIII),在病毒附着、膜融合、受体结合等方面发挥重要作用。EDI是EDII和EDIII的桥梁。EDII远端包含一个融合环(Fusion loop,FL),在pH依赖性构象变化期间插入宿主细胞体内膜推动膜融合。EDIII可以独立折叠为含有受体结合位点的蛋白。三个结构域均包含独立的抗原表位,但EDIII区域的表位更多且更具特异性,主要诱导型特异性中和抗体的产生,是较为理想的抗原表位。
然而,现有抗体虽然也能够有效的中和ZIKV病毒并特异性的结合至包膜蛋白结构域III,但其本质上仍为传统抗体,包含Fc段。传统的抗体在应用时具备以下局限性,①分子量大,组织和/或肿瘤穿透性差,不能穿过血脑屏障;②生产成本昂贵,改造复杂;③容易引发体内的炎症反应。ZIKV病毒感染后可引起严重的神经系统疾病,包括格林列巴综合征、新生儿小头畸形等,成功的穿过血脑屏障是治疗ZIKV感染引起的神经系统疾病的一大基础,已有研究证明,纳米抗体可穿过血脑屏障,表明纳米抗体在治疗神经系统疾病方面巨大的应用价值。
因此,开发针对ZIKV EDIII筛选中和性纳米抗体,为纳米抗体在治疗寨卡感染引起的临床疾病奠定了重要的基础。
发明内容
基于现有技术存在的问题,本发明旨在提供一种寨卡病毒中和性纳米抗体及其制备方法与应用。本发明筛选出的纳米抗体能够与ZIKV EDIII特异性结合,且分子量更小,纯度高,与传统抗体相比,本发明筛选出的纳米抗体能够识别更隐蔽的抗原位点,更具热稳定性、高溶解度、易表达、低免疫原性等优势,同时它保持了完整抗体序列的亲和力、特异性,在体外易于改造,对于寨卡病毒感染的预防或治疗具有重要应用价值。
基于上述目的,本发明采用的技术方案如下:
第一方面,本发明提供一种寨卡病毒中和性纳米抗体,所述纳米抗体特异性地结合至寨卡病毒包膜蛋白的结构域III(ZIKV EDIII),所述纳米抗体与寨卡病毒包膜蛋白的结构域III特异性结合的抗原表位的氨基酸序列如SEQ ID NO:1所示。
本发明筛选出的纳米抗体能够特异性识别寨卡病毒包膜蛋白结构域III上更为隐蔽的抗原位点,具有分子量小,仅由重链可变域组成,不含Fc段,但依然保持抗体序列的亲和力和特异性,对于寨卡病毒感染的预防或治疗具有重要意义。
优选地,所述纳米抗体具有SEQ ID NO:2或SEQ ID NO:3所示氨基酸序列。
第二方面,本发明提供一种分离的核酸,所述核酸为编码上述纳米抗体的核酸或其互补序列;所述核酸具有SEQ ID NO:4或SEQ ID NO:5所示核苷酸序列。
第三方面,本发明提供一种表达载体,所述表达载体包含上述核酸。
第四方面,本发明提供一种噬菌体,所述噬菌体包含上述的核酸或表达载体。
第五方面,本发明提供上述纳米抗体在制备预防或治疗寨卡病毒感染的药物中的应用。
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上述的纳米抗体以及其药物上可接受的佐剂。
第七方面,本发明提供一种制备上述纳米抗体的方法,包括如下步骤:
采集的健康羊驼的外周血分离出单核淋巴细胞,并提取单核淋巴细胞总RNA,逆转录成cDNA,利用特异性引物扩增重链抗体VHH基因片段,构建含有扩增纳米片段的噬菌体质粒库;
将所述噬菌体质粒电转化至感受态细胞,将VHH区片段展示在噬菌体表面,构建噬菌体展示文库;
以噬菌体展示文库为流动相,以ZIKV-EDIII作为靶抗原,孵育后洗去未结合的游离噬菌体,收集结合噬菌体并感染感受态细胞,经繁殖扩增后再次以噬菌体展示文库为流动相,以ZIKV-EDIII作为靶抗原进行孵育洗脱,重复洗脱三次,筛选出与靶蛋白ZIKV-EDIII具有亲和性的纳米抗体。
抗体的产生主要依赖于三种机制:①胚系细胞编码的抗体基因复杂多样,基因间可发生随机重组,形成不同种类的抗体分子;②B淋巴细胞发育成熟过程中,V-(D)-J基因重组引起抗体可变区的多样性,并且这种多样性不依赖抗原的刺激;③B淋巴细胞经抗原刺激后发生细胞突变,使抗体亲和力增加。本发明基于天然纳米抗体库无需免疫动物,节约时间、成本,利用文库的高度多样性允许文库中的序列识别任一潜在的抗原结合物,通过体外淘选富集技术,富集高特异性、高亲和力的抗体。本发明使用的天然纳米抗体库容量达2×109cfu,满足识别特异性纳米抗体的条件,本发明首次基于天然纳米抗体库,以ZIKV-EDIII作为靶抗原进行筛选得到与其具有较高亲和性、特异识别和结合能力的纳米抗体,相对于现有通过免疫驼类动物获得纳米抗体的方式相比,具有周期短、免疫原性低的优势。
优选地,特异性引物序列如SEQ ID NO:6和SEQ ID NO:7所示。
优选地,上述方法还包括对筛选的与靶蛋白ZIKV-EDIII具有亲和性的纳米抗体作进一步筛选的步骤,方法如下:
将筛选出的纳米抗体的核苷酸序列插入表达载体构建重组质粒,并导入菌体进行纳米抗体原核表达,经SELIA检测纳米抗体抗体的结合活性筛选、经噬斑减少中和实验鉴定获得与ZIKV-EDIII具有特异识别和结合能力的纳米抗体。
与现有技术相比,本发明的有益效果如下:
(1)本发明首次基于天然纳米抗体库,以ZIKV-EDIII作为靶抗原进行筛选得到与其具有较高亲和性、特异识别和结合能力的纳米抗体,相对于现有通过免疫驼类动物获得纳米抗体的方式相比,具有周期短、免疫原性低的优势。
(2)本发明筛选出的纳米抗体能够特异性识别寨卡病毒包膜蛋白结构域III上更为隐蔽的抗原位点,具有分子量小,仅由重链可变域组成,不含Fc段,但依然保持抗体序列的亲和力和特异性,对于寨卡病毒感染的预防或治疗具有重要意义
综上,目前针对ZIKV的抗体研究主要集中在鼠源或人源的单克隆抗体,尚未有中和性纳米抗体的研究,鼠源性单克隆抗体是免疫小鼠的B淋巴细胞与小鼠骨髓瘤细胞融合形成的杂交瘤细胞所分泌的抗体,生产过程复杂,且会产生严重的人抗鼠抗体反应,在应用中需要经过改造;人源的单克隆抗体往往分离自病人体内,且多靶向EDⅠ、EDⅡ,与登革病毒(DENV)间具有交叉反应性。本发明通过用ZIKV-EDⅢ从天然纳米抗体文库淘选特异性较高的中和性纳米抗体,最终筛选得到两株具有良好中和活性的纳米抗体,纳米抗体分子量小、免疫原性、稳定,在靶向治疗ZIKV感染中具有良好的发展前景;本发明筛选出的两株中和性纳米抗体Nb-E3和Nb-E5,特异性针对ZIKV-EDIII抗原,可利用原核表达系统进行大量表达,具备可溶性好、成本低及良好的生物活性,并经进一步验证可用于ZIKV感染的治疗及预防。
附图说明
图1为纳米抗体纯化后灰度定量图;
图2为纳米抗体识别ZIKV-EDIII ELISA图;
图3为SPR鉴定纳米抗体识别ZIKV-EDIII图;
图4为流式细胞术鉴定纳米抗体识别ZIKV/DENV图;
图5为qRT-PCR鉴定纳米抗体抑制ZIKV在细胞中复制能力显微镜镜检及上清中病毒载量图;
图6为噬斑减少中和实验测定纳米抗体中和能力图;
图7为肽段ELISA鉴定纳米抗体识别的潜在抗原表位。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例提供一种靶向ZIKV-EDⅢ的纳米抗体制备方法,包括如下步骤:
一、靶向ZIKV-EDⅢ蛋白纳米抗体的筛选及制备
1、纳米抗体天然噬菌体展示文库构建原理
本发明使用的天然纳米抗体文库来源于深圳康体生命科技有限公司,该天然文库共采集103只成年健康羊驼外周血细胞(PBMC),提取总RNA,逆转录成cDNA,利用特异性引物扩增重链抗体VHH基因片段,并转入噬菌体质粒pADL-10b中,构建含有扩增纳米片段的噬菌体质粒库,通过多次电转化至SS320感受态细胞,在辅助噬菌体M13K07的帮助下,将VHH区片段展示在噬菌体表面,获得羊驼天然噬菌体文库,抗体文库容量为2×109cfu,效价为1.28×1013pfu/mL。其中,特异性引物的序列如下:Forward:5’-3’:GGATCCATGGCGGTGCAGCCG(SEQID NO:6);Reverse primer:5’-3’:GAGCTCCGCACGCGGACTCCTC(和SEQ ID NO:7)。根据构建的天然文库来筛选能特异性结合ZIKV-EDⅢ抗原的纳米序列。
2、利用噬菌体展示技术淘选和富集ZIKV-EDⅢ纳米抗体
用CBS包被液(pH 7.6)包被50μg ZIKV-EDⅢ抗原(来源于本实验室),抗原菌株NCBI登陆号为KU740184.2,4℃包被过夜。次日用3%BSA封闭液于室温封闭2h,弃封闭液。每孔加入100μl噬菌体抗体库,室温孵育1h,弃去未结合的噬菌体,用含0.1%Tween-20的PBS清洗免疫管20次。每孔加入1ml 0.25mg/mL Trypsin溶液,室温洗脱30min。加入10μl 10%AEBSF终止洗脱液,收集免疫管中的溶液,即为第一轮筛选噬菌体洗脱液。将洗脱的噬菌体立即加入5ml OD600nm为0.5-0.6的SS320菌液中,37℃培养30min,涂布于2×YT-GA(终浓度100μg/ml AMP,2%葡萄糖)平板,37℃培养过夜。次日用涂布棒刮取过夜培养的全部菌落,取刮下的300μl细菌加入转接中至100ml 2×YT液体培养基中(含10μg/ml Tet和100μg/mlAMP),37℃,250rpm培养至菌液生长至OD600nm为0.5-0.6,加入辅助噬菌体M13K07(滴度为1013/ml)20μl,220rpm培养30min,分别加入终浓度为50μg/ml KAN和0.2mMIPTG,30℃,250rpm培养过夜。次日离心后,取上清加入20%PEG/2.5M NaCl沉淀噬菌体。收集得到第一轮噬菌体子文库,用于下一轮的筛选。共重复淘筛3次,使表面表达的纳米抗体噬菌体单克隆得到富集。
三轮筛选结果如下表1所示,第二轮筛选后效价为1014,相对于第一轮富集10倍,可直接用于三轮筛选。
表1 ZIKV-EDⅢ第一、二轮淘选噬菌体纯化效价
筛选轮数 | 输入效价 | 纯化效价 |
1 | <![CDATA[4.0×10<sup>13</sup>pfu/mL]]> | <![CDATA[1.2×10<sup>13</sup>pfu/mL]]> |
2 | <![CDATA[1.2×10<sup>13</sup>pfu/mL]]> | <![CDATA[8.0×10<sup>14</sup>pfu/mL]]> |
3、ZIKV-EDⅢPhage-ELISA检测阳性克隆
ELISA板包被1ng/μl ZIKV-EDⅢ抗原,同时平行包被BSA作对照,4℃包被过夜。挑取96个单克隆菌落至无菌96孔细胞板中,每孔加入200μl 2×YT液体培养基(含有100μg/mlAMP和10μg/ml Tet),37℃培养过夜,于次日取过夜菌液2μl转接至新的200μl 2×YT液体培养基(含有100μg/ml AMP和10μg/ml Tet)96孔细胞板中,37℃静置培养3-5h,加入辅助噬菌体M13K07,使噬菌体数:细菌个数达20:1,37℃静置培养30min,分别加入终浓度为50μg/mlKAN和0.2mM IPTG,30℃,250rpm培养过夜。于次日培养液离心后收集上清用来检测。取包被的ELISA板,每孔加入3%BSA室温封闭1h,室温清洗酶标板3次后,实验组和对照组每孔分别加入等量单克隆上清噬菌体,室温孵育2h。每孔加入200μl PBST共洗3次,加入M13Bacteriophage Antibody(HRP),室温1h。PBST洗5次,每孔加入TMB显色液,避光显色2-3min,加入1M HCl终止液,用酶标仪读取OD450nm值,当实验孔与BSA对照比值大于3,初步判为阳性孔,将阳性孔复测后仍为阳性孔的判定为阳性克隆。
4、阳性克隆基因序列分析
通过phage-ELISA、重复序列剔除后确定15株针对ZIKV-EDⅢ的纳米抗体,将其按照96孔板所在位置命名为Nb-A1、Nb-A7等。
二、CHIKV-E2纳米抗体可溶性原核表达及纯化
1、pET-SUMO原核表达载体质粒构建
将纳米抗体基因序列插入到pET-28a-SUMO表达载体的BamHI和XhoI酶切位点之间,使其与SUMO标签融合表达,SUMO的N端添加了6×His tag,可用于融合蛋白的纯化,委托上海捷瑞生物工程有限公司合成该质粒。
2、纳米抗体原核表达
取构建成功的重组表达质粒穿刺菌均匀涂布于含LB固体平板(KAN终浓度为50μg/mL),37℃培养过夜。挑单菌落扩大培养于含5mL的LB液体培养基的试管中,置于220rpm、37℃恒温摇床上过夜得种子菌;取种子菌液转接扩大培养于800mlLB液体培养基中,37℃220rpm振荡培养。待菌体OD600nm生长至0.6-0.8时,向菌液中加入终浓度为0.5mM IPTG于37℃诱导4h。12,000rpm离心10min弃上清,PBS重悬菌体,超声波细胞粉碎机进行裂解至清亮,12,000rpm 4℃离心10min,分别收集上清和菌体沉淀。
菌体破碎后沉淀即为包涵体,包涵体用包涵体溶解液(20mM Tris、5mM DTT、8MUrea)于4℃过夜溶解,12,000rpm/min离心5min,弃沉淀,上清用镍柱纯化后,洗脱液为含6M尿素的250mM的咪唑溶液,洗脱液与溶液A(20mM Tris-HCl,0.15M NaCl)等体积混合,加入透析袋中。将透析袋置于透析液中4℃磁力搅拌器中透析过夜。
3、纳米抗体Ni2+亲和层析柱纯化
融合蛋白亲和标签为His,利用HisPur Ni-NTA Resin(含Ni2+)纯化纳米抗体,取树脂1ml于亲和层析柱中,含10mM咪唑缓冲液平衡柱子,将包涵体溶后上清加入镍柱中,4℃孵育1h,2倍柱体积的洗脱液液(含25mM咪唑)洗去杂蛋白,最后用等体积的洗脱液(含250mM咪唑)洗脱目的蛋白,并收集洗脱液;采用超滤浓缩管(Millpore,3KD)对样品浓缩。15%SDS-PAGE检测蛋白纯化情况,结果如图1所示,图1A为Nb-C9、Nb-D8灰度定量,其中,1-5泳道:从左至右依次为0、2、4、6、8μg/μl;6-7泳道依次为Nb-C9、Nb-D8。图1B为Nb-E3、Nb-E5灰度定量。1-5泳道:从左至右依次为0、2、4、6、8μg/μl;6-7泳道依次为Nb-E3、Nb-E5。灰度定量结果显示Nb-E3蛋白和Nb-E5蛋白这两株纳米抗体具有较高的纯度,Nb-E3蛋白纯度为82.3%、Nb-E5蛋白纯度为92.1%。
三、ELISA检测纳米抗体结合活性
ELISA板包被ZIKV-EDⅢ(2μg/孔),4℃孵育过夜。于次日弃包被液,PBST(含0.05%Tween-20)洗板3次;3%BSA-PBST,37℃封闭1h;弃封闭液,PBST洗板3次;稀释抗体100倍(抗体稀释起始浓度为0.3μg/ml),37℃孵育1h;弃掉抗体稀释液,PBST洗板3次,每次5min;每孔加入1:1000稀释的SMT3-HRP单克隆抗体,37℃孵育1h;弃掉孔内液体,PBST洗板3次;TMB避光显色10min;2M H2SO4终止反应。结果判定:样品OD450nm值大于0.5为阳性,重复三次,取平均值。结果如图2所示,共得到4株阳性纳米抗体Nb-C9、Nb-D8、Nb-E3、Nb-E5。
四、Western Blot鉴定纳米抗体识别抗原线性表位
以ZIKV-EDⅢ、ZIKV裂解液、ZIKV灭活液对纯化后的纳米抗体进行Western blot验证。抗原经SDS-PAGE分离后转至PVDF膜上。2.5%脱脂奶粉室温封闭2h,以纯化后纳米抗体作为一抗,用封闭液稀释至1:1000,4℃孵育过夜,以1:1000稀释的HRP标记的SMT3-HRP,室温孵育1h;洗膜,ECL显影,曝光。结果如图3所示,本发明提供的两株纳米抗体对ZIKV具有特异的识别和结合能力,采用SPR技术分析了两株纳米抗体与ZIKV-EDⅢ结合亲和力和动力学参数,结果显示Nb-E3的解离常数为2.52nM;Nb-E5结合的解离常数为8.22Nm,表明两株纳米抗体可以与抗原结合。
五、流式细胞术鉴定纳米抗体特异性
ZIKV/DENV以MOI=1感染Vero细胞72h,4%多聚甲醛中固定细胞20min;1%脱模液透化细胞,每管加入纳米抗体3μg,4℃下孵育30min,使用Alexa Fluor 488抗6×His/鼠抗标记抗体在4℃下孵育30min。透膜液洗涤后,晾干,流式细胞仪检测识别结果,用商品化鼠抗ZIKV-E蛋白作阳性对照,结果如图4所示。
六、LSPR检测纳米抗体结合E2蛋白动力学特性
参照OpenSPRTM仪器说明安装COOH芯片。即以最大流速150μl/min运行,检测缓冲液为HEPES(PH=7.4)。信号基线平稳后,将200μl IPA上样,运行10s以排除气泡,基线平稳后,将样本环用缓冲液冲洗,空气排空气泡。信号达到基线值后,缓冲液流速调整为20μl/min。上样EDC/NHS(1:1)溶液激活芯片。将200μl配体溶液(激活缓冲液稀释的ZIKA EDIII)上样运行,时间为4min,再用HEPES(PH=7.4)冲洗样本环,并用空气排空。上样200μlBlocking溶液,缓冲液冲洗样本环,同样使用空气排空。观察基线5分钟,使基线平稳。以20μl/min的速度上样分析物(缓冲液稀释的Nbs),分析物、配体结合时间为设置为240s,自然解离360s。实验结果采用TraceDrawer(Ridgeview Instrumentsab,Sweden)软件分析,分析方法采用One To One模型。
七、噬斑减少中和实验与qRT-PCR
抗体按照30μg、3μg、0.3μg的梯度倍比稀释后与100μl MOI=0.1病毒等体积混和,37℃孵育1h。混合物接种单层Vero细胞,37℃吸附1h后移除接种物,加入2%FBS DMEM病毒维持液37℃、5%CO2培养,每日观察细胞病变,待细胞出现病变时,取上清,按照试剂盒(Omega,USA)说明书提取RNA。病毒载量标准曲线定量试剂盒(泽叶生物)测定,结果如图5和图6所示,共得到两株中和活性的纳米抗体序列Nb-E3、Nb-E5,其纳米抗体滴度分别为1:160、1:1280。
八、肽段ELISA鉴定纳米抗体与ZIKV-EDIII结合的潜在抗原表位
将ZIKV氨基酸序列按照15个氨基酸残基为一肽段,前后各重叠5个氨基酸的策略合成17个肽段并以一个不相关肽段为阴性对照共18个,送上海吉尔公司合成。将粉末状的抗原肽按照各自溶解性分别加入DMSO或ddH2O溶解,用漩涡振荡器充分混匀溶解后。每个抗原肽用包被液稀释为50ng/μl。抗原肽以2μg/孔的浓度包被96孔板4℃过夜。甩净孔中液体,加入250μl PBST溶液洗涤,共三遍。每孔加入200μl 2.5%BSA封闭液37℃封闭2h。弃去封闭液加入PBST洗三遍。抗体稀释1:100稀释度加入孔板中,每孔100μl,37℃孵育1h,弃去液体,加入PBST洗三遍。将HRP标记的鼠抗His抗体稀释为1:1000,每孔加入稀释抗体100μl,于37℃培养箱孵育1h。甩净孔中液体,加入PBST洗涤三遍。预先取出适量TMB显色液平衡至室温,每孔加入100μl,37℃避光孵育15min。每孔加入1M HCl终止液50μl,15min内在酶标仪中检测读数,Nb-E5肽段ELISA结果如图7所示,其能识别线性表位的氨基酸序列为:VGEKKITHHWHRS(SEQ ID NO:1),该表位是一个MHC II类分子识别的线性表位,可用作表位肽疫苗的设计。
综上,本发明所采用的天然纳米抗体文库共采集103只成年健康羊驼血样,分离了~1010数目的单核淋巴细胞(PBMC),利用巢式PCR技术从分离的PBMC扩增整套抗体重链可变区基因,克隆至噬菌体载体pADL-10b上,抗体以融合蛋白的形式表达在外壳表面库,容量为2×109cfu。以噬菌体展示文库为流动相,ZIKV-EDⅢ作为靶抗原,利用抗原-抗体特异性结合,孵育后洗去未结合的游离噬菌体,洗脱的结合噬菌体感染SS320感受态细胞后,经繁殖扩增,进行下一轮洗脱,经过3轮的生物淘选后,即可得到与靶蛋白ZIKV-EDⅢ具有亲和性的纳米抗体。经噬菌体ELISA进行初步筛选筛选,获取目的单克隆序列,将阳性克隆基因插入pET-28a-SUMO表达载体的BamHI、XhoI酶切位点之间。经噬斑减少中和实验鉴定,共得到两株中和活性的纳米抗体序列Nb-E3、Nb-E5。
其中,纳米抗体Nb-E3的氨基酸序列为:
MAVQLVESGGGLVQPGGSLRLSCKVSGRPFTTYALAWFRQAPGKEREFLTVGAWRGQIFTADHVRGRFTISLDTAKNTFDLQMNSLNIEDTAVYYCAAGSRSANTPQADPKSFDYWGQGTQVTVSSGAR(SEQ ID NO:2);
纳米抗体Nb-E3的核苷酸序列为:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGCCTGGAGGCTCTCTGAGACTCTCCTGTAAAGTCTCTGGACGCCCCTTCACTACATACGCCCTGGCCTGGTTCCGTCAGGCTCCAGGAAAGGAGCGTGAGTTCTTAACAGTAGGTGCCTGGCGTGGTCAAATATTTACGGCAGATCACGTGAGGGGCCGATTCACCATCTCCCTGGACACCGCCAAGAACACGTTCGATCTCCAAATGAACAGCCTGAACATTGAGGACACGGCCGTCTATTACTGTGCAGCAGGGAGTCGTTCAGCTAATACCCCGCAAGCCGACCCAAAGAGTTTTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGCGCACGCG(SEQ ID NO:4)。
纳米抗体Nb-E5的氨基酸序列为:
MAVQLVESGGGLVQPGGSLRLSCKVSGRPFTTYALGWFRQAPGKEREFLTVGAWRGQIFTAESVRGRFTISLDTAKNTFDLQMNSLNIEDTAVYYCAAGSRSSNTPQADPGSFDYWGQGTQVTVSSGAR(SEQ ID NO:3)。
纳米抗体Nb-E5的核苷酸序列为:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGCCTGGAGGCTCTCTGAGACTCTCCTGTAAAGTCTCTGGACGCCCCTTCACTACATATGCCCTGGGCTGGTTCCGTCAGGCTCCAGGGAAGGAGCGTGAGTTCTTAACAGTAGGTGCCTGGCGTGGTCAAATATTTACGGCAGAGTCCGTGAGGGGCCGATTCACCATCTCCCTGGACACCGCCAAGAACACGTTCGATCTCCAAATGAACAGCCTGAACATTGAGGACACGGCCGTTTATTACTGCGCAGCAGGAAGTCGTTCATCTAATACCCCGCAAGCCGACCCTGGGAGTTTCGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGCGCACGCG(SEQ ID NO:5)。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种寨卡病毒中和性纳米抗体,其特征在于,所述纳米抗体特异性地结合至寨卡病毒包膜蛋白的结构域III,所述纳米抗体与寨卡病毒包膜蛋白的结构域III特异性结合的抗原表位的氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述寨卡病毒中和性纳米抗体,其特征在于,所述纳米抗体具有SEQ IDNO:2或SEQ ID NO:3所示氨基酸序列。
3.一种分离的核酸,其特征在于,所述核酸为编码权利要求2所述纳米抗体的核酸或其互补序列;所述核酸具有SEQ ID NO:4或SEQ ID NO:5所示核苷酸序列。
4.一种表达载体,其特征在于,所述表达载体包含权利要求3所述的核酸。
5.一种噬菌体,其特征在于,所述噬菌体包含权利要求3所述的核酸或权利要求4所述的表达载体。
6.权利要求1或2所述纳米抗体在制备预防或治疗寨卡病毒感染的药物中的应用。
7.一种药物组合物,其特征在于,所述药物组合物包括权利要求1或2所述的纳米抗体以及其药物上可接受的佐剂。
8.一种制备权利要求1或2所述纳米抗体的方法,其特征在于,包括如下步骤:
采集的健康羊驼的外周血分离出单核淋巴细胞,并提取单核淋巴细胞总RNA,逆转录成cDNA,利用特异性引物扩增重链抗体VHH基因片段,构建含有扩增纳米片段的噬菌体质粒库;
将所述噬菌体质粒电转化至感受态细胞,将VHH区片段展示在噬菌体表面,构建噬菌体展示文库;
以噬菌体展示文库为流动相,以ZIKV-EDIII作为靶抗原,孵育后洗去未结合的游离噬菌体,收集结合噬菌体并感染感受态细胞,经繁殖扩增后再次以噬菌体展示文库为流动相,以ZIKV-EDIII作为靶抗原进行孵育洗脱,重复洗脱三次,筛选出与靶蛋白ZIKV-EDIII具有亲和性的纳米抗体。
9.如权利要求8所述方法,其特征在于,所述特异性引物序列如SEQ ID NO:6和SEQ IDNO:7所示。
10.如权利要求8所述方法,其特征在于,所述方法还包括对筛选的与靶蛋白ZIKV-EDIII具有亲和性的纳米抗体作进一步筛选的步骤,方法如下:
将筛选出的纳米抗体的核苷酸序列插入表达载体构建重组质粒,并导入菌体进行纳米抗体原核表达,经SELIA检测纳米抗体抗体的结合活性筛选、经噬斑减少中和实验鉴定获得与ZIKV-EDIII具有特异识别和结合能力的纳米抗体。
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