CN109627297B - 来自寨卡病毒e蛋白的中和表位及其应用 - Google Patents
来自寨卡病毒e蛋白的中和表位及其应用 Download PDFInfo
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- CN109627297B CN109627297B CN201811636781.8A CN201811636781A CN109627297B CN 109627297 B CN109627297 B CN 109627297B CN 201811636781 A CN201811636781 A CN 201811636781A CN 109627297 B CN109627297 B CN 109627297B
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Abstract
本发明公开来自寨卡病毒E蛋白的中和表位及其应用,表位肽是来源于寨卡病毒的E蛋白的片段,且至少包含E蛋白的第381‑395位和/或393‑420位氨基酸残基,且所述变体与其所源自的表位肽仅在于氨基酸残基的保守置换,且保留了其所源自的表位肽的生物学功能;所述E蛋白第381‑395位氨基酸残基序列为SEQ ID NO:1,所述E蛋白第393‑420位氨基酸残基序列为SEQ ID NO:2;本发明提供了针对寨卡病毒E蛋白的中和表位及应用。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及来自寨卡病毒E蛋白的中和表位及其应用。
背景技术
寨卡病毒(Zika virus,ZIKV)是一种经蚊媒传播的黄病毒属病毒,于1947年首次从乌干达恒河猴体内分离得到。之后,ZIKV一直沉默数十年,从未发生过大流行,仅偶尔出现个别感染病例,症状轻微,表现为发热、头疼、肌痛、皮疹等类似普通流行性感冒症状。自2007年开始,ZIKV流行不断爆发,给流行地区带来了重大经济负担和严重的健康威胁。2015年底,ZIKV开始出现在巴西地区,之后迅速在全球播散,蔓延至近百个国家和地区。同时,流行地区出现了ZIKV引发的婴儿小头症以及成年人吉兰-巴雷综合征。在巴西,对于感染ZIKV的孕妇,超声检查显示42%的胎儿出现发育不全,最终可能会导致胎儿小头症、胎儿先天性畸形甚至胎儿死亡等严重的临床结果。此次ZIKV传播速度快、流行范围广且引发的症状严重,引发了全球范围的密切关注。除此之外,研究还发现ZIKV感染还会损伤男性生殖系统,可能会导致男性不育。总的来说,ZIKV感染在全球范围内引发了严重的公共卫生健康安全威胁。
2016年2月,WHO将ZIKV感染及其所引发的中枢神经系统病变列为国际突发公共卫生事件,并呼吁全球共同努力,携手研发针对ZIKV感染的预防疫苗和治疗药物。WHO统计目前已有45个备选疫苗处于研发中,其中部分已进入临床试验阶段,但还没有ZIKV预防疫苗和治疗药物上市。目前,防蚊灭蚊等消灭传播途径的方法是预防ZIKV传播的主要措施,对症治疗是主要的治疗措施,但疗效并不尽人如意。因此,针对ZIKV有效的预防疫苗和治疗药物急需研发。
ZIKV包膜蛋白E蛋白在病毒结合并进入宿主细胞过程中发挥着关键作用,因此E蛋白是ZIKV研发疫苗的重要靶点。E蛋白作为病毒的重要组成部分,具有很强的免疫原性,是激发机体针对病毒发生免疫反应的重要成分。因此,ZIKV E蛋白可能含有许多中和表位,刺激机体产生针对中和表位的中和性抗体。当前,以病毒中和性抗体为基础的的抗体治疗已经应用在许多疾病上。因此,发现ZIKV的中和表位及针对这些表位的有效中和性抗体对治疗ZIKV感染具有重大的意义。同时,在研发中还应该避免黄病毒属病毒可能会出现的抗体依赖性增强感染效应(Antibody-dependent enhancement,ADE)。
发明内容
针对上述技术问题,本发明提供了针对寨卡病毒E蛋白的中和表位及应用。
一种分离的表位肽或其变体,所述表位肽是来源于寨卡病毒的E蛋白的片段,且至少包含E蛋白的第381-395位或393-420位氨基酸残基,且所述变体与其所源自的表位肽仅在于氨基酸残基的保守置换,且保留了其所源自的表位肽的生物学功能;
所述E蛋白第381-395位氨基酸残基序列为SEQ ID NO:1,
所述E蛋白第393-420位氨基酸残基序列为SEQ ID NO:2;
SEQ ID NO:1Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu LysLys;
SEQ ID NO:2Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser ThrIle Gly Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Arg Arg。
一种表位肽偶联物,包含本发明提供的表位肽或其变体和与之偶联的偶联部分。
优选地,所述偶联部分为放射性核素、药物、毒素、细胞因子、酶、荧光素、载体、脂类和生物素中的一种或多种组合。
优选地,所述偶联部分通过选自下述的偶联方法偶联至表位肽或其变体:MBS法、戊二醛法、活泼酯法、碳二亚胺法、卤代硝基苯法及亚氨酸酯法。
一种蛋白疫苗,包括本发明所述的表位肽或其变体,以及药学上可接受的载体和/或赋形剂。
优选地,所述表位肽可以是单独的或串联的、偶联至其他蛋白的或不偶联至其他蛋白的。
优选地,所述蛋白疫苗为检测用组合物,其用于检测寨卡病毒抗体(特别是中和性抗体)的效价。
优选地,所述蛋白疫苗为用于制备抗寨卡病毒的抗血清或抗体的组合物。
一种多克隆抗体或其抗原结合片段,所述多克隆抗体能够特异性结合本发明所述的表位肽或其变体。
优选地,所述多克隆抗体为中和性多克隆抗体。
一种抗体偶联物,包含本发明提供的多克隆抗体或其抗原结合片段,以及与之偶联的偶联部分。
优选地,所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体、脂类和生物素中的一种或多种;
优选地,所述偶联部分通过选自下述的偶联方法偶联至表位肽:MBS法、戊二醛法、活泼酯法、碳二亚胺法、卤代硝基苯法及亚氨酸酯法。
本发明所述的表位肽或其变体、肽偶联物、蛋白疫苗、多克隆抗体或其抗原结合片段,在制备预防寨卡病毒感染的疫苗组合物中的应用。
本发明所述的表位肽或其变体、肽偶联物、蛋白疫苗、多克隆抗体或其抗原结合片段,在制备预防和或治疗寨卡病毒感染的药物中的应用。
优选地,所述多克隆抗体的使用量不小于12.59μg/mL;进一步优选地,能够特异性结合SEQ ID NO:1的多克隆抗体的使用量不小于30.58μg/mL;能够特异性结合SEQ ID NO:2的多克隆抗体的使用量不小于23.57μg/mL。
一种用于检测寨卡病毒的ELISA试剂盒,包括:用于包被酶标记抗原的检测板、稀释液、洗涤液、显色液、终止液、酶标记抗原;所述酶标记抗原为本发明所述的表位肽或其变体。
用于在受试者(优选哺乳动物,例如人)中治疗和/或预防ZIKV感染与所述感染相关的一种或多种疾病或病症的方法,其包括,给有此需要的受试者施用治疗和/预防有效量的本发明提供的表位肽、表位肽偶联物、多克隆抗体或其抗原结合片段、抗体偶联物。
制备抗寨卡病毒的抗血清或抗体的方法,所述方法包括:
1)给非人动物施用有效量的本发明的表位肽、或本发明的表位肽的偶联物,以在所述动物体内诱发抗寨卡病毒的免疫应答;
2)从所述动物分离抗寨卡病毒的抗血清
3)从所述抗血清中分离抗体。
制备抗寨卡病毒的抗血清或抗体的方法,所述方法包括:
1)从所述动物分离能够产生抗寨卡病毒抗体的B细胞;
2)将所述B细胞与骨髓杂交瘤细胞融合,以产生能够分泌抗寨卡病毒抗体的杂交瘤细胞;
3)分离和培养所述杂交瘤细胞;
4)从所述杂交瘤细胞的培养物中分离抗寨卡病毒抗体。
筛选本发明的中和表位的方法为:灭活ZIKV免疫兔子后获得ZIKV免疫后血清,用此血清对ZIKV E蛋白线性多肽库通过ELISA实验进行筛选,得到2个免疫显性表位(优势表位)肽。另外,通过生物信息学对2个优势表位的分析,发现了附近的另一个潜在中和表位。将2个优势表位肽和1个潜在中和表位肽偶联至柱材,从兔ZIKV免疫后血清中提取针对这3个表位肽的多克隆抗体。其中,1个优势表位肽和1个预测的潜在中和表位肽的兔多克隆抗体具有ZIKV中和活性,因此,这两个表位是来自ZIKV E蛋白的线性中和表位。
综上所述,本发明提供了来自ZIKV E蛋白的2个线性中和性表位,以及获得线性中和性表位的方法;本发明提供了针对上述线性中和表位的中和性抗体,以及获得中和性抗体的方法;本发明提供了中和性抗体在用于治疗ZIKV感染中的用途;本发明还提供了能够特异性结合本发明表位肽的多克隆抗体的有效剂量。
与现有技术相比,本发明的技术方案具有以下优点:本发明提供了来自ZIKV E蛋白的中和表位及针对其的中和性抗体。所述的中和性表位为进一步研发有效的ZIKV多肽疫苗提供了广阔前景。所述的中和性抗体为临床上ZIKV感染的治疗提供一类安全有效的治疗抗体。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例1中的兔ZIKV免疫后血清对ZIKV的空斑中和实验结果;
图2是本发明实施例2中的兔ZIKV免疫后血清与ZIKV E蛋白线性多肽库的酶联免疫吸附试验反应结果;
图3是本发明实施例3中的针对优势表位P1(E蛋白第331-345位氨基酸)和P2(E蛋白第381-395位氨基酸)以及预测潜在中和表位肽P3(E蛋白第393-420位氨基酸)的兔多克隆抗体对ZIKV的空斑中和实验结果;
图4是本发明实施例4中的针对中和表位P2(E蛋白第381-395位氨基酸)和P3(E蛋白第393-420位氨基酸)的兔多克隆抗体抑制ZIKV感染的浓度梯度依赖性结果。
具体实施方式
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
实施例1获取ZIKV免疫后兔血清及其中和性验证
本实施例获取ZIKV免疫后兔血清及其中和性验证的方法包括以下步骤:
1、BHK21细胞使用含10%FBS DMEM培养基在37℃、5%CO2条件下培养,待其在T75培养瓶中长成单层,弃细胞培养液,向每个T75中加入500μL ZIKV/10mL稀释液(DMEM培养基),在37℃、5%CO2条件下吸附2h,随后弃病毒感染液,加入20mL维持液(含2%FBS DMEM培养基),每日观察细胞,待细胞病变达70%以上时,收集感染上清。
2、在Beckman超速离心机SW41离心管底部加5mL 20%蔗糖,再加30mL感染上清,25000rpm、4℃超速离心2.5h,收集离心管底部沉淀,用PBS重悬。向重悬病毒液中加入终浓度为0.05%的甲醛,常温放置7天灭活ZIKV。使用BCA试剂盒检测灭活ZIKV浓度。
3、选用2kg左右的新西兰兔,免疫灭活ZIKV 50μg/只,免疫剂量1ml/只(铝佐剂500μL),免疫间隔2周,第3次免疫后1周取血,获得ZIKV免疫后兔血清。
4、将BHK21细胞按2*106/孔铺入12孔板,在37℃、5%CO2条件下培养24小时,待细胞长成无间隙单层时用于检测免疫后兔血清对ZIKV感染的抑制活性实验。
5、将ZIKV免疫后血清在56℃水浴30min后,用DMEM培养基将其按1:20稀释。将等体积的血清稀释液和ZIKV感染液(30PFU/孔)混合37℃孵育1h。兔免疫前血清操作同上,作为对照。
6、将免疫后血清和病毒孵育的混合液加入孔板,37℃、5%CO2条件下吸附2h,为实验组。免疫前血清与病毒孵育的混合液为对照组。不加血清只有病毒的设为阳性对照组,不加血清也不加病毒的为阴性对照孔。每组均设3孔。
7、弃去感染液,加入1%低熔点琼脂(含2%FBS),在37℃、5%CO2条件下培养6天左右,加入1g/mL结晶紫(含4%甲醛)染色12h,水流冲去琼脂,即可计算实验结果。
结果如图1所示,A表示免疫前血清、B表示免疫后血清、C表示阳性对照、D表示阴性对照;根据图1可知灭活ZIKV免疫后兔血清能够有效的中和ZIKV,抑制其感染。说明上述免疫方法可以成功免疫兔子,免疫后血清可以用来筛选ZIKV E蛋白的优势表位。
实施例2ZIKV E蛋白优势表位筛选
1、将来自ZIKV毒株SZ01的E蛋白的504个氨基酸序列按照长15个氨基酸、重复10个氨基酸的规则合成100个多肽。
2、将上述多肽混在包被液(PH 9.6的NaHCO3溶液)中,按照10μg/mL浓度50μL/孔加入ELISA板中,无关肽按照10μg/mL同样条件包被,灭活ZIKV按照1μg/mL。每个肽或灭活病毒包被6个孔。4℃过夜。
3、弃去多肽包被液,洗液(0.05%PBST)洗一遍,甩干。
4、加2%脱脂牛奶100μL/孔封闭,室温放置2h。洗液洗三遍,甩干。
5、兔免疫前血清和免疫后血清均稀释1:100,每个肽或灭活病毒所包被6个孔中,3个分别加稀释后免疫前血清和免疫后血清,50μL/孔。室温放置1h。洗液洗三遍,甩干。
6、HRP酶标兔抗鼠二抗1:2500稀释加入各孔,50μL/孔。室温放置1h。洗液洗四遍,甩干。
7、TMB显色液显色,用1M H2SO4终止反应后,使用酶标仪检测每孔的反应强度。
8、通过分析ELISA实验结果,筛选ZIKV E蛋白优势表位P1和P2。再通过对优势表位进行生物信息学分析,预测ZIKV E蛋白中和表位P3。
结果如图2所示,ZIKV E蛋白第331-345位氨基酸序列P1(SEQ ID NO:3,Gln TyrAla Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met)和第381-395位氨基酸序列P2(SEQ ID NO:1,Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys)与兔免疫后血清有很明显的反应,是ZIKV的免疫显性表位(优势表位)。通过生物信息学分析,ZIKV E蛋白第393-420氨基酸序列P3(SEQ ID NO:2,Glu Lys Lys Ile Thr His His TrpHis Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly Ala ArgArg)也可能是ZIKV的中和表位肽。
本实施例建立了ZIKV E蛋白全覆盖高容量的多肽抗原库,即将来自ZIKV毒株SZ01的E蛋白的504个氨基酸序列按照长15个氨基酸、重复10个氨基酸的规则合成100个多肽,作为筛选的多肽库。用灭活的ZIKV免疫3次兔子,每次免疫间隔2周,即可获得兔ZIKV免疫后血清。将兔ZIKV免疫后血清与ZIKV做空斑中和实验;用以确认ZIKV免疫兔子的效果,结果显示兔ZIKV免疫后血清能够有效中和ZIKV感染,说明ZIKV成功免疫兔子,产生了针对ZIKV的中和性抗体。将来自ZIKV E蛋白100个抗原多肽包被在ELISA反应板上,以兔ZIKV免疫后血清作为反应一抗,以羊抗兔抗体作为反应二抗进行ELISA反应,结果发现有多肽P1(E蛋白第331-345位氨基酸)和P2(E蛋白第381-395位氨基酸)与兔免疫后血清反应最强。因此发现了ZIKV E蛋白的免疫显性表位(优势表位)P1和P2。还通过生物信息学的方法对2个优势表位进行分析,发现P2附近还有一个区域P3(E蛋白第393-420位氨基酸)是潜在的ZIKV E蛋白中和表位。
实施例3针对表位肽的多克隆抗体对ZIKV毒株SZ01的空斑中和实验
(1)将BHK21细胞按2*106/孔铺入12孔板,在37℃、5%CO2条件下培养24小时,待细胞长成无间隙单层时用于检测多克隆抗体对ZIKV毒株SZ01的空斑中和实验。
(2)多克隆抗体用PBS稀释成100μg/mL,与ZIKV感染液(30PFU/孔)等体积混合,在37℃、5%CO2条件下孵育1小时。免疫前血清IgG同样处理作为对照。
(3)将多抗和免疫前血清IgG与ZIKV混合液加入各孔感染BHK21,在37℃、5%CO2条件下吸附2小时。实验组和对照组每组各3孔。
(4)弃去感染液,加入1%低熔点琼脂(含2%FBS),在37℃、5%CO2条件下培养6天左右,加入1g/mL结晶紫(含4%甲醛)染色12h,水流冲去琼脂,即可计算实验结果。
结果如图3所示,A表示P1多克隆抗体实验结果、B表示P2多克隆抗体实验结果、C表示P3多克隆抗体实验结果、D表示免疫前实验结果、E表示阳性对照实验结果、F表示阴性对照实验结果;根据图3可知,针对P2和P3的多克隆抗体能够有效中和ZIKV,而P1多克隆抗体则没有中和活性(图3)。因此,P2(E蛋白第381-395位氨基酸)和P3(E蛋白第393-420位氨基酸)是来自ZIKV E蛋白的线性中和表位肽。
本实施例将P1、P2和P3多肽分别偶联至柱材(SulfoLink Coupling Resin)上,按照柱材说明书操作,分别提取了针对所述3个表位肽的兔多克隆抗体。再通过空斑中和实验检测抗体是否能够抑制ZIKV感染:如果针对该表位肽的多克隆抗体能够中和ZIKV,说明该表位是来自ZIKV E蛋白的中和表位;反之,则说明该表位仅仅是来自ZIKV E蛋白的优势表位,而非中和表位。结果发现针对P2和P3的多克隆抗体均能有效抑制ZIKV感染,而针对P1的多克隆抗体则没有ZIKV中和作用。因此,P2和P3即为来自ZIKV E蛋白的中和表位。
实施例4中和性抗体抑制ZIKV毒株SZ01的浓度梯度依赖性
(1)将BHK21细胞按2*106/孔铺入12孔板,在37℃、5%CO2条件下培养24小时,待细胞长成无间隙单层时用于检测多克隆抗体对ZIKV毒株SZ01的空斑中和实验。
(2)将P2和P3以及免疫前血清IgG做2倍的倍比稀释,浓度分别为100μg/mL、50μg/mL、25μg/mL、0.78μg/mL。再与ZIKV感染液(30PFU/孔)等体积混合。在37℃、5%CO2条件下孵育1小时。
(3)将上述混合液加入各孔感染细胞,在37℃、5%CO2条件下吸附2小时。每组均有3孔。
(4)弃去感染液,加入1%低熔点琼脂(含2%FBS),在37℃、5%CO2条件下培养6天左右,加入1g/mL结晶紫(含4%甲醛)染色12h,水流冲去琼脂,即可计算实验结果。
结果如图4所述,显示针对P2和P3的多克隆抗体能够有效中和ZIKV,且具有浓度梯度依赖性。P2多抗中和ZIKV的IC 50为30.58μg/mL,P3多抗中和ZIKV的IC 50为23.57μg/mL。
本实施例对上述中和表位P2和P3的中和性多抗进行系列梯度稀释,再分别检测对ZIKV感染的抑制率,结果显示针对P2的中和性多抗的IC50为30.58μg/mL;针对P3的中和性多抗的IC50为23.57μg/mL。因此,针对上述中和表位的中和性抗体能够有效抑制ZIKV感染。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
序列表
<110> 复旦大学
<120> 来自寨卡病毒E蛋白的中和表位及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys
1 5 10 15
<210> 2
<211> 28
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser Thr Ile Gly
1 5 10 15
Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Arg Arg
20 25
<210> 3
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met
1 5 10 15
Claims (2)
1.一种分离的表位肽,其特征在于,所述表位肽是来源于寨卡病毒的E蛋白的片段,其氨基酸序列为SEQ ID NO:1、SEQ ID NO:2。
2.权利要求1所述的表位肽在筛选寨卡病毒中和抗体中的应用。
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