CN108640981A - H7n9亚型流感病毒重组蛋白及病毒疫苗 - Google Patents
H7n9亚型流感病毒重组蛋白及病毒疫苗 Download PDFInfo
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Abstract
本发明提供编码鸭源H7N9亚型流感病毒HA的重组表达载体，生产重组蛋白的方法以及应用重组蛋白的方法。重组表达的H7HA具有良好的生物活性，为禽流感疫苗对珍稀禽类保护性研究提供了新的方法。
Description
技术领域
本发明提供H7N9亚型流感病毒HA蛋白的重组表达载体，重组蛋白的表达方法以及使用重组蛋白的方法。
背景技术
流感病毒能够引起严重的人兽共患病，宿主范围包括禽类、哺乳动物如猪、犬、猫、虎、熊猫、牛、海豹和鲸鱼等以及人类，可引发禽流感、季节性流感疫情。自2013年年初以来，通过《国际卫生条例》报告途径获知，迄今报告了总共1564例人感染甲型H7N9禽流感病毒实验室确诊病例。
流感病毒名称中的Hx和Nx分别来自两种蛋白：表面糖蛋白血凝素(Hemagglutinin， HA)和神经氨酸酶(Neuraminidase，NA)。HA蛋白有助于流感病毒结合宿主细胞，而NA蛋白则是病毒复制后从细胞出芽的必要条件。病毒内部序列有8个基因片段，8个基因之间可以重新进行组合，这一过程称之为重组(reassortment，也称为重配)。到目前为止，已经发现的A型流感病毒(Influenza A virus，IAV)包括有18种HA亚型和11种NA亚型(BodewesR,ZohariS,KrogJ S,et al.Spatiotemporal analysis of the geneticdiversity of seal influenza A(H10N7)virus，Northwestern Europe[J].J Virol,2016,90(9):4269-4277；Nayak D P,Hui E K,Barman S.Assembly and budding ofinfluenza virus[J].Virus research,2004,106(2):147-165)。
在全球范围内，禽感染H7亚型是很普遍的，但严重的人感染却很少见。H7亚型禽流感病毒可分为两个不同的基因谱系：北美谱系和欧亚谱系。
中国CDC国家流感中心(the Chinese National Influenza Center,CNIC)对H7N9禽流感病毒基因数据进行基因组比对和亲缘关系分析后发现，在H7N9病毒的8个基因片段中，H7HA片段与A/duck/Zhejiang/12/2011(H7N 3)密切相关，因此推测HA可能来源于浙江鸭群中的禽流感病毒，属于禽流感病毒欧亚谱系，并可追溯至东亚地区野鸟中分离的相似病毒；N9NA片段与A/wildbird /Korea/A14/2011(H7N9)密切相关，推测可能来源于沿着东亚候鸟迁徙途径迁徙的韩国野鸟。其余6个内部基因片段(PB2、PB1、PA、NP、M、NS) 来源于中国上海、浙江、江苏等地的鸡群中的H9N2禽流感病毒，这些病毒与北京燕雀A /brambling/Beijing/16/2012(H9N2)密切相关。中国科学院微生物研究所一个流感研究团队认为H7N9禽流感病毒可能起源于多重基因重组, 从事这种研究是为了了解这类变异有多大可能会发生自然演变并引起在人类中的大流行，包括对病毒的传播能力，以及耐药性方面的研究工作。
由H7N9流感病毒造成的人死亡病例使得有必要研制疫苗来预防H7N9流感。
目前，在流感病毒编码的蛋白中，以HA蛋白的研究最为深入，HA蛋白在决定流感病毒抗原性、病毒侵入宿主细胞等方面起作用，NA蛋白则帮助成熟的子代病毒从宿主细胞表面释放(Spackman E,Swayne D.E.Vaccination of gallinaceous poultry forH5N1highly pathogenic avian influenza: current questions and new technology[J].Virus Res.2013.178(1):121–132)。流感病毒的HA上存在抗原决定簇，抗原决定簇是一些能激发宿主产生抗流感病毒抗体的氨基酸区域，抗原决定簇内氨基酸位点发生变化可导致流感病毒的抗原性发生改变。HA蛋白是宿主获得性免疫系统识别的主要对象，因而成为疫苗研制的主要靶标。
美国Novavax公司的科研人员构建了重组H7N9流感疫苗，该疫苗包括未经修饰的HA蛋白和NA蛋白来自A/Anhui/1/2013株，以及克隆入杆状病毒载体的M1蛋白(来自 A/Indonesia/05/2005株)；感染昆虫细胞的杆状病毒可分泌由HA、NA和M1构成的病毒样颗粒。结果显示，H7N9流感病毒样颗粒疫苗对同种病毒的血凝抑制(HI)抗体滴度在1∶64 以上，且能与异种H7N3流感病毒发生交叉HI反应；同样，不同剂量的H7N9流感VLP疫苗均能诱导机体产生抗NA抗体，非同源H7亚型流感疫苗能与H7N3、H7N9流感病毒发生血凝抑制反应，而不能诱导产生抗N9亚型流感病毒NA抗体。(Vaccine，2013，31(40))。
在国内，目前禽流感的防控中广泛使用的疫苗主要是全病毒灭活疫苗，该疫苗抗原成分全，免疫原性很好，但也存在灭活不全，依赖于鸡胚生产，干扰流行病学的监测等缺点，且不断有新亚型毒株出现，需要不断更新种毒株。另外对于珍稀动物和禽类的防疫，需要开发新的疫苗策略(王祥,周东明.基于血凝素(HA)的新型流感疫苗研究进展[J].生命科学,2014,26(09):943-948；张文俊，薛涛，吴小伟，等.鸭源H5亚型禽流感全禽源分子标记疫苗候选株的构建[J].畜牧兽医学报,2011,45(5):685-691；张妍，张文俊，李群辉，等.Clade2.3.2H5N1亚型禽流感疫苗候选株的构建[J].畜牧兽医学报,2012,43 (6):915-921)。已有研究表明，在细菌、昆虫细胞、哺乳动物细胞等多种表达系统中表达的HA蛋白均有活性，利用HA蛋白研制基因工程疫苗在防控禽流感中发挥着重要的作用
发明内容
本发明提供编码鸭源H7N9亚型流感病毒HA的重组表达载体，生产重组蛋白的方法以及应用重组蛋白的方法。
本发明以鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)HA基因为模板，在昆虫细胞中重组h7‐HA蛋白，该重组蛋白具有天然H7N9的HA蛋白的活性，能够形成重组杆状病毒颗粒。
本发明进一步提供包括编码HA的重组DNA分子的组合物，该重组DNA分子可以进一步是具有90％同源性的重组DNA分子，优选具有95％同源性的重组DNA分子，进一步优选具有98％同源性的重组DNA分子，所述重组DNA分子组装的重组蛋白具有相似的天然H7N9 的HA三聚体构象及活性，能够形成重组杆状病毒颗粒。
本发明进一步提供包括HA重组蛋白的组合物，该HA重组蛋白可单独存在也可与其他重组蛋白结合，所述HA重组蛋白可以是重组蛋白的片段，所述片段含有H7N9的HA三聚体构象及活性，该组合物进一步含有药学可接受的载体或形成疫苗所需的相应佐剂。包含该重组蛋白、蛋白片段或病毒颗粒的药学上有用的组合物可按照已知方法制备，如混合一种药学上可接受的载体或佐剂而制备，这种组合物包含的重组蛋白、蛋白片段或病毒颗粒应足够引起希望的生物学或药学效应。
进一步，本发明提供针对HA重组蛋白或HA重组蛋白片段或重组病毒颗粒的抗体。
进一步，本发明提供一种试剂盒，所述试剂盒包含含有HA重组蛋白和HA重组蛋白片段中的至少一种的免疫原性组合物。
进一步，本发明提供的工艺包括重组蛋白的生产工艺以及重组蛋白的施用工艺。所述重组蛋白的生产工艺包含用鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)HA基因为模板，转染昆虫细胞，在允许编码重组体蛋白的DNA表达的条件下培养转染的昆虫细胞，并提纯重组体蛋白。施用重组蛋白包括HA重组体蛋白或其片段组合物或重组病毒颗粒用于禽类，包括野生禽类和畜养禽类。
本发明的治疗性或预防性或诊断性组合物以足够治疗、预防或诊断H7N9感染的量施用于个体，所述组合物可以通过多种途径如皮下、局部、口服、经粘膜或肌肉内适用于个体。
本发明的疫苗包含足够在宿主细胞中诱导中和抗体形成所需的抗原决定簇的HA重组蛋白或其片段或组装的病毒颗粒，这种疫苗足够安全不具有毒副作用，可通过一种有效途径施用，与疫苗载体相容，制剂稳定。该疫苗可以是液体制剂或混悬剂或胶囊剂，可以单剂量或多剂量用药。
本发明点的方法使防止H7N9感染的亚病毒疫苗配方成为可能，施用该方法，可制备单价或多价H7N9疫苗，如仅含有鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)的HA重组蛋白、蛋白片段或病毒颗粒的单价疫苗，或通过混合来自不同病毒亚型或不同来源的同种病毒亚型的HA重组蛋白或NA重组蛋白或其组合而制备多价疫苗。
本发明所述的由HA重组蛋白或HA重组蛋白片段或重组病毒颗粒产生相应的抗体，所述抗体包括单克隆抗体和多克隆抗体，以及其片段如Fv，Fab片段，它们都能结合抗原或半抗原。
本发明所述的HA重组蛋白，病毒颗粒和相应的抗体可用于对H7N9感染进行血清分型和H7N9筛查，将重组蛋白、病毒颗粒或抗体置于试剂盒的配方中，这种试剂盒可通过间隔的形式装有载体，适合放置于至少一种容器中，该试剂盒中进一步含有试剂，如检测标记抗原或酶底物的工具等。
HA蛋白是AIV的主要抗原蛋白，在病毒的致病力、感染性以及免疫保护中起着重要作用。本发明中选取杆状病毒表达系统对我国自主分离的一株目前鸭源低致病性的H7‐HA进行了研究，将合成的HA基因克隆至PFastBac1载体上，构建了由GP67信号肽、HA胞外区、Thtombin酶切位点、三聚体标签及His标签构成的复合载体，利用sf9昆虫细胞及High5 获得分泌表达具有生物活性的纯度90％以上的HA蛋白。通过Western-blot、间接免疫荧光分析表明，具有保留较好的反应原性。本发明进行了昆虫表达系统中高表达量、高纯度载体构建实现了H7N9型禽流感病毒HA抗原基因在杆状病毒表达系统中成功表达，表达的蛋白可以作为亚单位疫苗和诊断试剂的抗原。本发明通过SPF鸡的免疫试验，进一步验证该抗原蛋白的免疫效果和安全性，为禽流感疫苗对珍稀禽类保护性研究提供新的方法。
H7N9亚型流感病毒中HA蛋白在决定流感病毒抗原性、病毒侵入宿主细胞等方面起作用，NA蛋白则帮助成熟的子代病毒从宿主细胞表面释放，针对本发明的鸭源流感病毒 A/Duck/Fujian/S4170/2014(H7N9)，基于HA重组蛋白的抗体或疫苗免疫原性更为有益，较多抗原如基于HA与NA重组蛋白的抗体或疫苗的免疫效力更为突出。
附图说明
图1：显示的鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)HA基因PCR扩增结果。
图2：显示重组杆状病毒颗粒rBacmid‐H7HA的PCR鉴定结果。
图3：显示纯化H7‐HA重组蛋白SDS‐PAGE检测及Western blot鉴定。
图4：通过IFA检测H7‐HA蛋白在感染的SF9细胞中的表达
图5：H7‐HA蛋白免疫后鸡血清抗体效价。
具体实施方式
通过以下实施例以进一步说明本发明，而不限制本发明于这些实施例的个别事例。
用于下列实施例的材料如下：
毒株、载体、试剂及实验动物A/Duck/Fujian/S4170/2014株H7N9亚型AIV由中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室分离、鉴定和保存；pFastBac1载体、pMD18T-HA(含有H7N9病毒HA基因开放阅读框)和sf9昆虫细胞、High Five细胞本实验室保存；各种限制性内切酶、昆虫细胞培养基及CellFectin II转染试剂盒均购自 Invitrogen公司。H7N9亚型的阳性血清购自哈尔滨维科生物技术开发公司。抗His单克隆抗体购自武汉三鹰。NiSepharose excel树脂购自GE公司。Alexaflour 488标记的山羊抗鸡LgY购自abcam公司，Anti-Chicken IgG(H+L)Antibody,DyLight 800-Labeled购自KPL公司。3周龄SPF鸡由哈尔滨兽医研究所实验动物中心提供。
实施例1：
HA基因的扩增及重组供体质粒的构建
根据禽流感病毒A/Duck/Fujian/S4170/2014(H7N9)HA基因的序列运用Primer软件设计引物扩增HA抗原基因，上游引物P1：
5'ATAAGAATGCGGCCGCTTATGGGATTCACATACAGCGGGATAAGA 3'。
下游引物P2：
5'CCCTCGAGTTAGTGATGATGATGATGATGTCC 3'。
重组杆状病毒颗粒鉴定的通用引物为M13(M13F：5′-GTTTTTCCCAGTCACGAC-3′；M13R:5′-CAG-GAAACAGCTATGAC-3′)，引物由吉林库美生物科技有限公司合成。以 pMD18T-HA为模板，扩增H7N9的HA基因，回收纯化目的条带。将目的片段和pFastBac1 载体分别进行NotI/XhoI双酶切，回收纯化后连接，热激法转化DH5，感受态细胞，筛选阳性克隆，提取重组供体质粒pFastH7-HA，并进行酶切测序验证。
HA扩增及重组Bacmid‐H7HA的PCR鉴定，以pMD18T-HA(含有H7N9病毒HA基因开放阅读框)为模板，HAF/HAR为引物，通过PCR扩增重组质粒得到约1500bp的片段(图1)，大小与预期相符。用M13F/M13R鉴定结果表明重组杆状病毒颗粒Bacmid‐H7HA构建正确(图 2)。
实施例2：
HA重组穿梭质粒的构建与鉴定
将pFastH7-HA供体质粒转化至DH10Bac感受态细胞，在含有卡那霉素(50μg/mL)，庆大霉素(7μg/mL)，四环(10μg/mL)，X-gal(100μg/mL)和IPTG(40μg/mL) 的LB平板上培养48h，蓝白斑筛选，选取白色菌落。抽提DNA，以M13通用引物进行PCR检测，筛选阳性克隆杆状病毒颗粒，命名为BacmidH7-HA。并提取BacmidH7-HA重组杆状病毒颗粒，用于细胞转染。
实施例3：
重组杆状病毒的获得与扩增
参照CellFectinII转染试剂说明书，将BacmidH7-HA转染生长状态良好的sf9昆虫细胞，27℃恒温培养箱培养36～72h，待出现细胞病变后，收集上清作为第一代重组病毒。将细胞上清在sf9昆虫细胞传4代，收集第4代细胞上清，以1：4的比例加入P4 代重组杆状病毒，即200mLP4代病毒加入800mL的HighFive细胞中，27℃摇床旋转培养，转速120rpm/min,获得HA蛋白浓度较高的细胞培养上清。
实施例4：
Western-blot鉴定HA蛋白的表达
将重组病毒rBacH7-HA接种sf9昆虫细胞，感染72h后，收集培养上清，用于Western-blot分析。以1∶200稀释的H7N9阳性血清为一抗，以DyLight 800标记的山羊抗鸡IgG作为二抗，进行检测；以同样方法处理的正常sf9昆虫细胞蛋白样品作为阴性对照。将重组质粒转染sf9昆虫细胞，HA以分泌可溶形式表达；利用Ni Sepharose excel 树脂亲和层析纯化重组蛋白，重组蛋白经SDS-PAGE电泳检测，大小约为65KD，与预期结果一致。蛋白纯度达到90％以上，SDS-PAGE电泳结果未见明显杂蛋白条带(图3)。
实施例5：
HA蛋白的间接免疫荧光鉴定
将重组病毒Bacmid‐H7HA接种于生长状况良好的sf9昆虫细胞，感染4h后，用PBST洗涤3次。用预冷的甲醇室温固定10min，自然干燥。一抗选用1∶100稀释的H7N9 阳性血清，37℃孵育1h，用PBST洗涤3次。二抗选用1∶500稀释的Alexa flour 488标记的山羊抗鸡LgY，37℃孵育1h，用PBST洗涤3次，置于荧光显微镜下观察结果。以同样方法处理的sf9细胞为阴性对照。
rBacHA杆状病毒感染sf9昆虫细胞，进行免疫荧光检测后，在荧光显微镜下观察显示， rBacHA感染的sf9细胞出现绿色荧光，而正常的sf9细胞和阴性对照均未出现荧光，表明H7N9的HA基因在sf9昆虫细胞中获得正确表达(图4)。
实施例6：
SPF鸡免疫保护实验
取3周龄SPF鸡20只，每组10只，分别为免疫组和阴性对照组，一免使用弗氏完全佐剂乳化疫苗H7HA，分别胸肌多点注射免疫组，蛋白终浓度为100μg/ml，免疫剂量为0.2mL/羽，即20μg/羽；一免后两周使用H7HA蛋白加强免疫，免疫剂量为0.15mL/羽，即15μg/ 羽。
首次免疫后，每隔一周进行采血，分离血清，通过血凝抑制试验测定抗体效价水平，结果见图5。H7-HA蛋白免疫后，HI抗体滴度逐渐升高，两周加免后，HI抗体滴度进一步升高，免疫后4周可达5log2以上(图5)。
实施例7：
病毒滴度测定
HA和HI检测方法根据国家标准(GB/T-18936-2003)进行。将采集SPF鸡喉头及泄殖腔拭子按照10倍倍比稀释，每个稀释度接种3枚10日龄的SPF鸡胚，37℃培养48h后检测各稀释度血凝阳性鸡胚数目，根据Reed-Muench方法计算EID50。
SPF鸡的免疫攻毒实验表明，与阴性对照相比，HA蛋白免疫组能够对 A-duck-Fujian-S4170-2014(H7N9)毒株产生100％保护力，而非免疫对照组SPF鸡在攻毒后全部排毒(表1)。
表1 H7HA免疫后SPF鸡攻击A/Duck/Fujian/S4170/2014(H7N9)保护性试验
Claims (10)
1.一种分离和纯化的H7N9亚型流感病毒的HA重组蛋白及其功能衍生物，所述流感病毒是鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)。
2.如权利要求1所述的HA重组蛋白及其功能衍生物，所述功能衍生物进一步为HA重组蛋白的片段，所述片段含有H7N9的HA三聚体活性构型区域。
3.如权利要求1或2所述的HA重组蛋白及其功能衍生物，所述功能衍生物进一步为具有90％同源性的重组DNA分子，优选具有95％同源性的重组DNA分子，进一步优选具有98％同源性的重组DNA分子，所述重组DNA分子组装的重组蛋白具有相似的天然H7N9的HA三聚体活性构型区域。
4.如权利要求1‐3任一项所述的HA重组蛋白及其功能衍生物，该蛋白由一种合成系统生产，此系统包括一种重组体宿主，优选重组体宿主为昆虫细胞。
5.由权利要求1‐3任一项所述的HA重组蛋白及其功能衍生物构成的病毒样颗粒。
6.包含权利要求1‐5任一项所述的HA重组蛋白及其功能衍生物或由其组装的病毒样颗粒的组合物，所述组合物进一步含有药学可接受的载体或形成疫苗所需的相应佐剂。
7.如权利要求6所述组合物，该组合物进一步含有来自不同病毒亚型或不同来源的同种病毒亚型的HA重组蛋白或NA重组蛋白及其功能衍生物。
8.如权利要求6或7所述的组合物，该组合物进一步制备为疫苗或诊断试剂盒，所述试剂盒可用于对H7N9感染进行血清分型和H7N9筛查。
9.由权利要求1‐5任一项所述的HA重组蛋白及其功能衍生物或由其组装的病毒样颗粒产生的抗体，所述抗体包括单克隆抗体和多克隆抗体，以及其片段如Fv，Fab片段，它们都能结合抗原或半抗原。
10.一种生产权利要求1所述的HA重组蛋白及其功能衍生物的方法，该方法包括
(1)、以鸭源流感病毒A/Duck/Fujian/S4170/2014(H7N9)HA基因为模板，将其克隆到昆虫细胞表达载体pFastBac1中，
(2)、构建重组质粒rpFBacH7HA，转化DH10Bac感受态细胞提取重组Bacmid‐H7HA。
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