CN111358943A - 一种新冠病毒的双靶向免疫增强型多价疫苗及其制备方法 - Google Patents
一种新冠病毒的双靶向免疫增强型多价疫苗及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种新冠病毒的双靶向免疫增强型多价疫苗及其制备方法,该疫苗为nCoV多价表位肽与ACE‑2基因重组表达载体的复合物;nCoV1多价表位肽/nCoV2多价表位肽/nCoV3多价表位肽分别由表位nCoV480‑493/nCoV499‑512/nCoV120‑133与穿膜序列WYSPKLWMR、表位nCoV 586‑600/表位nCoV 128‑142/表位nCoV 420‑434和接头序列组成,穿膜序列位于氨基端,表位nCoV 586‑600/表位nCoV 128‑142/表位nCoV 420‑434序列位于羧基端,表位与穿膜序列或表位与表位之间以接头序列连接;该疫苗可成通过穿膜序列进入细胞,有效促进新冠病毒抗原表位nCoV1/nCoV2/nCoV3进入抗原递呈途径激发特异性B细胞和CD4+T细胞应答,同时ACE2基因在细胞内表达生成ACE2蛋白,刺激机体产生ACE2抗体,从而封闭细胞ACE2蛋白,达到阻断病毒通过S蛋白结合ACE2蛋白的作用。
Description
技术领域
本发明涉及一种多价疫苗,特别涉及一种新冠病毒的双靶向免疫增强型多价疫苗及该疫苗的制备方法。
背景技术
2019年新型冠状病毒(2019 novel Coronavirus,2019-nCoV)的流行规模迅速增大,其大规模感染对人民的生活造成极大的影响。目前2019-nCoV引起的新冠肺炎病例已在全球多个国家发现,世界卫生组织(WHO)宣布2019-nCoV列为国际关注的突发公共卫生事件。新型冠状病毒的基因组编码了刺突蛋白(Spike protein:S)、包膜蛋白(Envelopeprotein:E)、膜蛋白(Membrane glycoprotein:M)和核衣壳蛋白(Nucleocapsid protein:N)等结构蛋白,刺突蛋白介导与宿主的受体结合和膜融合。
由于冠状病毒灭活疫苗或减毒疫苗在正常人体应用时存在潜在的危险性,因此分子量小、安全性高的多肽疫苗是新型疫苗研究的重点。而研究多肽疫苗的第一步是从新型冠状病毒的基因序列中找出有免疫原性的抗原表位或抗原决定簇。现代疫苗理论认为,最有效的疫苗需要包含B细胞和CD4+T细胞抗原表位,从而既能够有效诱导体液免疫。鉴于重要靶点S蛋白在病毒侵入细胞中的重要作用,一些S蛋白已经获得了有效的疫苗。
ACE2由805个氨基酸组成,是具有单一胞外催化结构域的I型跨膜糖蛋白。人类ACE2基因已经被克隆并被定位到X染色体上。像ACE一样,ACE2有两个结构域:氨基末端催化结构域和羧基末端结构域。ACE2主要在心脏、肾脏和睾丸中定位,通常定位于上皮细胞的腔面。冠状病毒通过表达ACE2的细胞腔面进行感染时,其感染效力提高10倍。冠状病毒以Clathrin蛋白依赖性方式与ACE2结合并内在化,以使其进入细胞。膜融合是通过蛋白酶Spike介导激活,病毒RNA被释放到细胞质中,从而引发冠状病毒感染。
由于细胞膜的屏障作用,生物大分子不能自由进入细胞。近年来,一些具有细胞膜穿透能力的小分子多肽相继被发现,其可有效携带外源性大分子进入细胞,并对宿主细胞没有显著毒副作用。这些具有细胞穿透能力的多肽被命名为细胞穿膜肽(CPP)。研究发现,WYSPKLWMR可有效促进与之偶联的外源表位进入MHC-Ⅰ类抗原递呈途径,表现为动力学显著增强,且该过程表现为蛋白酶体/TAP非依赖、氨基肽酶依赖。
发明内容
有鉴于此,本发明的目的之一在于提供一种新冠病毒的免疫增强型多价疫苗,目的之二在于提供所述新冠病毒的免疫增强型多价疫苗的制备方法。
为达到上述目的,本发明采用如下技术方案:
新冠病毒的免疫增强型多价疫苗,其特征在于:该疫苗为nCoV1多价表位肽、nCoV2多价表位肽、nCoV3多价表位肽与ACE2基因重组表达载体的的复合物;
所述nCoV1多价表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV480-493:QHTDINFTATASFG、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV586-600:YTSYTIVGALYVTWS组成;
所述nCoV2多价表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV499-512:CKPHQVNLSLNGNT、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV128-142:NLLFTEQLGAPLGIT组成
所述nCoV2表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV120-133:SSSFDCIVNLLFTE、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV 420-434:VDVLVNVSATKIQNL组成
在所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽中,穿膜序列均位于氨基端,CD4细胞表位序列均位于羧基端,表位与穿膜序列或表位之间均以接头序列连接。
所述接头序列为AYAY。
所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
所述ACE2基因重组表达载体是在真核表达载体pcDNA3.1中插入ACE2序列而得到。
向溶解有ACE2基因重组表达载体的0.9%的KCl溶液中滴加溶解nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的水溶液,滴加完毕后继续搅拌60分钟,再静置60分钟,即得。
一种用于制备所述新冠病毒的免疫增强型多价疫苗的制备方法,其特征在于:所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
本发明的有益效果在于:本发明疫苗可通过穿膜序列进入细胞,有效促进新冠病毒抗原表位nCoV1/nCoV2/nCoV3进入抗原递呈途径激发特异性B细胞和CD4+T细胞应答,同时ACE2基因在细胞内表达生成ACE2蛋白,刺激机体产生ACE2抗体,从而封闭细胞ACE2蛋白,达到阻断病毒通过S蛋白结合ACE2蛋白的作用。动物实验结果证实,本发明疫苗能够激发特异性的B细胞和CD4+T细胞应答,分泌S抗体和ACE2抗体,达到预防病毒感染机体的作用。本发明疫苗制备方法简单,成本低廉,在新冠病毒预防领域有着较好的开发应用前景。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步的详细描述,其中:
图1为nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的一级结构示意图;
图2为ACE2基因的琼脂糖凝胶电泳鉴定图;
图3为ACE2重组真核表达质粒双酶切产物电泳鉴定图;
图4为本发明疫苗的透射电镜图;
图5为酶联免疫(ELISA)法检测表位肽可与患者血清特异性结合;
图6为酶联免疫(ELISA)法检测本发明疫苗诱导的特性行抗体;
具体实施方式
以下将参照附图,对本发明的优选实施例进行详细的描述。优选实施例中未注明具体条件的实验方法,通常按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著,黄培堂等译,科学出版社,2002年)中所述的条件,或按照制造厂商所建议的条件。
一、新冠病毒的免疫增强型多价疫苗的制备
1、nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的设计与合成
本发明设计的nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的一级结构如图1所示,各表位与穿膜序列或表位肽序列之间以接头序列连接,以便于各表位保持独立性和能够有效递呈。在本实施例中,接头序列为丙氨酸-酪氨酸-丙氨酸-酪氨酸(Ala-Tyr-Ala-Tyr,AYAY);nCoV1表位肽的氨基酸序列如SEQ ID No.1所示,nCoV2表位肽的氨基酸序列如SEQID No.2所示,nCoV3表位肽的氨基酸序列如SEQ ID No.3所示;同时,以卵清蛋白(OVA)表位肽作为对照肽(由表位OVA257-264、穿膜序列HIV-Tat49-57、内质网滞留信号序列KDEL和接头序列组成,其氨基酸序列如SEQ ID No.4所示。
多肽的合成在ABI 431A型固相多肽合成仪(美国PE公司)上进行。方法采用标准芴甲氧羰基(Fmoc)方案,精氨酸采用两次偶联。起始选用0.125mmol对羟甲基苯氧甲基聚苯乙烯树脂(HMP树脂),按照多肽序列使肽链从羧基端逐个向氨基端延伸,每种氨基酸的用量为0.5mmol,与树脂的摩尔比为4:1。各种氨基酸的α-氨基为Fmoc保护,其余侧链保护基团分别为:Lys(Boc)、Ser(tBu)、Glu(OtBu)、Arg(Pmc)、His(Trt)、Thr(tBu)和Tyr(tBu)。第一个氨基酸连接到树脂上用4-二甲氨基吡啶(DMAP),氨基酸的活化用1-羟基苯并三唑(HOBt)和二环己基碳二亚胺(DCC),偶联后用体积分数为20%的哌啶水溶液去除Fmoc保护基。多肽合成后,将树脂-粗肽产品在冰浴条件下混合于10mL切割液A(由结晶苯酚0.75g、1,2-乙二硫醇即EDT 0.25mL、苯甲硫醚0.5mL、去离子水0.25mL和三氟乙酸即TFA10mL组成)中,待切割液温度上升至室温后,搅拌反应2小时,使肽链从树脂上裂解下来,同时去除多种保护基团。将反应混合液经G4玻砂漏斗过滤以去除树脂,先后用1mL TFA、5~10mL二氯甲烷反复冲洗反应瓶、树脂和漏斗。将滤液在常温低压下蒸发至1~2mL,加入50mL预冷乙醚沉淀多肽,4℃放置过夜,G6玻砂漏斗过滤,真空抽干,即得多肽粗品,-20℃保存备用。
将多肽粗品用二甲基亚砜(DMSO)溶解制成浓度为20mg/mL的溶液,经孔径为0.45μm的微孔滤膜过滤后,在AKTA explorer 100型中压液相色谱仪(瑞典Amerssham Bioscienc公司)上用SOURCE凝胶柱进行纯化。流动相A由体积百分含量为10%的乙醇和体积百分含量为0.1%的TFA组成,流动相B由体积百分含量为90%的乙醇和体积百分含量为0.1%的TFA组成;洗脱梯度为:先用流动相A 1.5个柱体积洗脱,再用流动相A和流动相B的混合液(流动相B占混合液的体积分数在8个柱体积内由0%逐渐增加至80%)洗脱,再用流动相A和流动相B的混合液(流动相B占混合液的体积分数在0.5个柱体积内由80%逐渐增加至100%)洗脱,在主峰处收集多肽溶液,冷冻干燥,即得多肽纯品,用DMSO溶解,-20℃保存备用。
将多肽纯品用Delta 600型高压液相色谱仪(美国Waters公司)鉴定纯度,采用Symmetry ShieldTM C18柱,流动相由体积百分含量为10%~60%的乙腈和体积百分含量为0.1%的TFA组成,梯度洗脱,流速为1mL/min。结果显示,合成多肽的纯度均达到90%以上。同时,将多肽纯品用API 2000LC/MS型电喷雾离子化质谱仪测定分子量。结果显示,合成多肽的分子量均与理论值相符。
2、ACE2基因重组表达载体的构建
(1)ACE2全长编码基因的克隆
根据GenBank登录号为NC_000023.11的ACE2基因序列,设计并合成上下游引物,序列如SEQ ID No.5和SEQ ID No.6所示。PCR引物以扩增ACE2全长编码基因;以人胎盘cDNA文库为模板进行PCR,PCR条件为:94℃预变性3分钟,然后94℃变性30秒、56℃退火30秒,72℃延伸30秒,共30个循环,最后72℃延伸5分钟;PCR产物经琼脂糖凝胶电泳鉴定、凝胶回收试剂盒切胶回收纯化后,与载体pGEM-T连接,连接产物转化大肠杆菌JM109感受态细胞,用含有Amp/IPTG/X-GAL的培养基进行蓝白斑筛选,挑取白斑培养,提取质粒,委托上海生工公司测定基因序列,将序列正确的阳性克隆质粒命名为pGEM-T/ACE2;
PCR产物的琼脂糖凝胶电泳鉴定图如图2所示,其中M泳道为DNA分子量标准,1泳道为PCR产物,可见1泳道在约2400bp处呈单一特异性条带,与预期结果相符;质粒的测序结果显示插入基因序列与ACE2全长编码基因序列一致。
(2)ACE2基因重组真核表达载体的制备
根据ACE2全长编码基因序列和真核表达载体pcDNA3.1的多克隆位点,设计并合成PCR引物,序列如SEQ ID No.7和SEQ ID No.8所示;以扩增包含ACE2全长编码基因、5’端XBI酶切位点和3’端Ecor1酶切位点的cDNA片段;以pGEM-T/ACE2为模板进行PCR,PCR条件与步骤(1)相同;PCR产物经琼脂糖凝胶电泳鉴定、凝胶回收试剂盒切胶回收纯化后,用限制性内切酶XBI和Ecor1进行双酶切,双酶切产物经凝胶回收试剂盒切胶回收纯化后,与同样经XBI和Ecor1双酶切的pcDNA3.0在T4 DNA连接酶的作用下进行连接,连接产物转化大肠杆菌TOP10感受态细胞,用含有Amp/IPTG/X-GAL的培养基进行蓝白斑筛选,挑取白斑培养,提取质粒,用XBI和Ecor1进行双酶切鉴定,并委托上海生工公司测定基因序列,将序列和阅读框架正确的阳性克隆质粒命名为pcDNA3.1/ACE2;
重组真核表达质粒双酶切产物的琼脂糖凝胶电泳鉴定图如图3所示,其中M泳道为DNA分子量标准,1泳道为双酶切产物,可见1泳道在约3500bp和2400bp处出现两条电泳带,与预期结果相符;质粒的测序结果显示插入基因序列无突变,阅读框架正确,无移码。
3、nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与ACE2基因重组表达载体的复合物的制备
将pcDNA3.0/ACE2用浓度为0.9%的KCl溶解制成浓度为500μg/mL的溶液,取该溶液100μL,在混旋状态下以5μL/min的速度滴加总浓度为1000μg/mL的多肽溶液(由摩尔比为1:1:1的nCoV1表位肽、nCoV2表位肽、nCoV3表位肽组成)100μL,滴加完毕后继续混旋60分钟,再静置60分钟,即得nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与ACE2基因重组表达载体的复合物,也即本发明所述新冠病毒的免疫增强型多价疫苗。
透射电镜分析:将新制复合物滴于200目铜网上,吸附3分钟,用吸水纸吸干,晾干30秒,以质量体积百分浓度为1%的醋酸铀水溶液负染30秒,用吸水纸吸干,晾干30秒,80kV透射电镜观察。结果如图4所示,所得复合物呈大小均一的近圆形颗粒,绝大多数颗粒长径小于25nm。
二、ELISA法检测表位肽与患者血清特异性结合
采用ELISA检测试剂盒进行检测,按照试剂盒说明书操作:在96孔培养板中,每孔加入体积百分浓度为70%的乙醇溶液100μL,室温放置10分钟,PBS洗涤,再加入总浓度为1000μg/mL的多肽溶液(由摩尔比为1:1:1的nCoV1表位肽、nCoV2表位肽、nCoV3表位肽组成)100μL,温度4℃孵育过夜,PBS洗涤,再加入质量百分浓度为2%的脱脂奶粉溶液100μL,室温封闭2小时,PBS洗涤,再加入新冠病毒患者血清100μL,温度37℃孵育48小时,PBST(即含有质量百分浓度为0.1%的吐温20的PBS)洗涤,再加入生物素标记的羊抗人抗体(稀释度为1∶100)100μL,温度37℃孵育1.5小时,PBST洗涤,再加入链霉亲和素标记的碱性磷酸酶(稀释度为1∶5000)100μL,温度37℃孵育1小时,PBST洗涤,拍干培养板,再加入即用型BCIP/NBT底物反应液100μL,室温避光显色2~10分钟,用H2SO4终止反应,同时设置阴性对照组,酶联仪检测各实验组的OD值。
结果如图5所示,实验组的OD值明显高于对照组、空白组,表明本发明的多价表位可以特异性结合患者体内抗体。
三、新冠病毒的免疫增强型多价疫苗的免疫原性研究
将30只6-8周龄雌性Babl/c小鼠随机分为3组:实验组、对照组和空白组,每组10只;实验组以浓度为0.5mg/mL的本发明疫苗为免疫原,对照组以浓度为0.5mg/mL的OVA表位肽与pcDNA3.0/ACE2复合物为免疫原,空白组以浓度为0.1mol/L、pH为7.4的PBS为免疫原;各组于小鼠背侧尾根部皮下注射免疫原,每只100μL,之后每间隔1周,以同样方法加强免疫1次,共免疫3次。免疫后一周,取Babl/c小鼠的尾静脉全血,分离血清。ELISA法检测本发明疫苗刺激小鼠生成抗体的能力。
采用ELISA检测试剂盒进行检测,按照试剂盒说明书操作:在96孔培养板中,每孔加入体积百分浓度为70%的乙醇溶液100μL,室温放置10分钟,PBS洗涤,再加入总浓度为1000μg/mL的多肽溶液(由摩尔比为1:1:1的nCoV1表位肽、nCoV2表位肽、nCoV3表位肽组成)100μL,温度4℃孵育过夜,PBS洗涤,再加入质量百分浓度为2%的脱脂奶粉溶液100μL,室温封闭2小时,PBS洗涤,再加入小鼠血清100μL,温度37℃孵育48小时,PBST(即含有质量百分浓度为0.1%的吐温20的PBS)洗涤,再加入生物素标记的羊抗鼠抗体(稀释度为1∶100)100μL,温度37℃孵育1.5小时,PBST洗涤,再加入链霉亲和素标记的碱性磷酸酶(稀释度为1∶5000)100μL,温度37℃孵育1小时,PBST洗涤,拍干培养板,再加入即用型BCIP/NBT底物反应液100μL,室温避光显色2~10分钟,用H2SO4终止反应,同时设置阴性对照组,酶联仪检测各实验组的OD值。
结果如图6所示,实验组的OD值明显高于对照组、空白组,表明本发明的疫苗复合物具有良好的免疫原性,能够有效刺激机体产生高效价抗体。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
序列表
<110> 重庆医科大学附属永川医院
<120> 一种新冠病毒的双靶向免疫增强型多价疫苗及其制备方法
<141> 2020-03-02
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Claims (6)
1.新冠病毒的免疫增强型多价疫苗,其特征在于:该疫苗为nCoV1多价表位肽、nCoV2多价表位肽、nCoV3多价表位肽与ACE2基因重组表达载体的的复合物;
所述nCoV1多价表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV 480-493:QHTDINFTATASFG、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV 586-600:YTSYTIVGALYVTWS组成;
所述nCoV2多价表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV 499-512:CKPHQVNLSLNGNT、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV 128-142:NLLFTEQLGAPLGIT组成
所述nCoV2表位肽由穿膜序列WYSPKLWMR、新冠病毒S蛋白B细胞表位nCoV 120-133:SSSFDCIVNLLFTE、接头序列:AYAY、新冠病毒S蛋白CD4细胞表位nCoV 420-434:VDVLVNVSATKIQNL组成
在所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽中,穿膜序列均位于氨基端,CD4细胞表位序列均位于羧基端,表位与穿膜序列或表位之间均以接头序列连接。
2.根据权利要求1所述新冠病毒的免疫增强型多价疫苗,其特征在于:所述接头序列为AYAY。
3.根据权利要求1所述新冠病毒的免疫增强型多价疫苗,其特征在于:所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
4.根据权利要求1所述新冠病毒的免疫增强型多价疫苗,其特征在于:所述ACE2基因重组表达载体是在真核表达载体pcDNA3.1中插入ACE2序列而得到。
5.一种用于制备权利要求1所述新冠病毒的免疫增强型多价疫苗的制备方法,其特征在于:在搅拌条件下,向溶解有ACE2基因重组表达载体的0.9%的KCl溶液中滴加溶解nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的水溶液,滴加完毕后继续搅拌60分钟,再静置60分钟,即得。
6.根据权利要求5所述用于制备新冠病毒的免疫增强型多价疫苗的制备方法,其特征在于:所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
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