CN113372454A - 尼帕病毒受体结合糖蛋白及其应用 - Google Patents
尼帕病毒受体结合糖蛋白及其应用 Download PDFInfo
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Abstract
本发明涉及生物医药领域,特别涉及尼帕病毒受体结合糖蛋白及其应用。本发明以尼帕病毒受体结合糖蛋白(G)为研究对象,利用哺乳动物细胞对其进行可溶性表达,纯化并通过免疫小鼠制备特异性血清,并通过间接ELISA鉴定结合活性。结果表明:尼帕病毒G蛋白在Expi293F细胞中获得可溶性表达,纯化得到的目的蛋白大小正确,纯度大于99%;制备的抗血清能特异性结合目的蛋白。上述研究为尼帕病毒诊断与疫苗研究奠定了基础。
Description
技术领域
本发明涉及生物医药领域,特别涉及尼帕病毒受体结合糖蛋白及其应用。
背景技术
尼帕病毒病是一种由尼帕病毒(NipahVirus)引起的主要侵害中枢神经***和呼吸***的急性损伤,且高度致死的***共患传染病,该病首次1998年暴发于马来西亚,其病原为尼帕病毒(Nipahvirus,NiV),由于该病毒在尼帕双溪(Sungai Nipah)地区被分离到,因此得名。
尼帕病毒为单股负链RNA病毒,属于副黏病毒科(Paramyxoviridae)亨尼帕病毒属(Henipavirus)成员,与另一个1994年在澳大利亚布里斯班发现的享德拉病毒(HendraVirus,HeV)同属。由于对人致死率高(40%-70%),被列为生物安全4级病原。该病毒的主要自然宿主为狐蝠科(Pteropid)的果蝠(Fruitbat),猪、牛、马、山羊、猫、狗、鼠、兔、狐狸、掠鸟、八哥等也是尼帕病毒的天然宿主。果蝠活动范围较广,NiV有可能在一定区域内的果蝠中相互传播,因此,相同种类蝙蝠分布的国家和地区会面临病毒突变、疫病暴发流行的危险,自1998年首次发现NiV以来,NiV的流行主要分布在东南亚地区和西太平洋地区。但近几年,西太平洋地区NiV暴发的频率逐渐降低,而东南亚地区国家,尤其是孟加拉国地区NiV暴发非常频繁,更引起了全世界的关注,NiV在此类周边国家的流行态势对我国造成了潜在威胁。我国目前尚未检出NiV,但我国东南沿海地区和云南地区具有适合NiV流行的生态环境和宿主动物,且已发现NiV的自然宿主,在蝙蝠体内存在尼帕病毒抗体,提示我国存在尼帕病毒或者类似病毒的自然疫源地。因此,开展针对NiV综合防控关键技术的相关研究,具有重要的现实意义。
NiV病毒粒子具有多形性,有囊膜,病毒颗粒大多数呈现不规则的球状,直径大多数长约150-200nm,少数病毒颗粒呈现丝状体,长度可达10000nm。NiV主要有2个遗传谱系:NiV马来西亚株(NiV Malaysia,NiV-MY)和NiV孟加拉株(NiVBangladesh,NiV-BD)。其中NiV-MY株基因组18246个核苷酸,而NiV-BD株基因组18252个核苷酸。目前流行毒株主要是马来西亚株,比同属HeV多12个碱基,编码N(核蛋白)、P(磷蛋白)、M(基质蛋白)、F(融合蛋白)、G(受体结合蛋白)、L(聚合酶)等至少6种蛋白,其中N蛋白、P蛋白、L聚合酶一起称为复制复合体,结合在病毒RNA上,与病毒基因组的转录和复制有关,M蛋白参与维持病毒囊膜形态,F蛋白、G蛋白参与蛋白的融合和吸附,与病毒入侵有关。NiV在其包膜内包含两种膜锚定糖蛋白,G蛋白和F蛋白,其中G蛋白与宿主细胞膜蛋白ephrin-B2、ephrin-B3结合,而此两种蛋白在宿主细胞表面高度保守,所以目前F、G为抗病毒与疫苗研究的主要靶点,NiV G蛋白全长共602个氨基酸,属于II型跨膜糖蛋白,N末端位于膜内,膜内部分极短,疏水的跨膜结构域靠近N端,C末端位于膜外,由一个“球型”头部区域和较长的“外茎”区域组成。
因此,提供可在哺乳动物细胞中可溶性表达纯化尼帕病毒受体结合糖蛋白具有重要的现实意义。
发明内容
有鉴于此,本发明提供了尼帕病毒受体结合糖蛋白及其应用。本发明以尼帕病毒受体结合糖蛋白(sG)为研究对象,利用哺乳动物细胞对其进行可溶性表达,纯化并通过免疫小鼠制备特异性血清,以期为后续开展尼帕病毒的蛋白结构、入侵机制、诊断、疫苗及中和抗体等相关研究奠定基础。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了尼帕病毒受体结合糖蛋白,以尼帕病毒G蛋白为基础,保留氨基酸残基172-602aa位置,即球形头部结构域(图2A),删除细胞质尾部区、跨膜疏水区以及氨基酸残基71-171位置外茎区,命名为sG;同时,N段添加人组织纤溶酶原激活物信号肽(tPA)序列、Strep标签以及可切除标签的TEV蛋白酶酶切序列,两端分别添加酶切位点HindⅢ和BamHⅠ。
在本发明的一些具体实施方案中,所述尼帕病毒受体结合糖蛋白具有:
(I)、如SEQ ID No.1所示的氨基酸序列;或
(II)、如(I)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列,且与(I)所述的氨基酸序列功能相同的氨基酸序列;或
(III)、与如(I)或(II)所述的氨基酸序列具有90%以上同一性的氨基酸序列;
所述取代、缺失或添加一个或多个氨基酸中的4个或5个。
本发明还提供了编码所述尼帕病毒受体结合糖蛋白的核酸分子。
在本发明的一些具体实施方案中,所述核酸分子具有:
(Ⅰ)、如SEQ ID No.2所示的核苷酸序列;或
(Ⅱ)、如SEQ ID No.2所示的核苷酸序列的互补核苷酸序列;或
(Ⅲ)、与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质,但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的核苷酸序列;或
(Ⅳ)、与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列经取代、缺失或添加一个或两个核苷酸序列获得的核苷酸序列,且与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(V)、与(Ⅰ)、(Ⅱ)、(Ⅲ)或(Ⅳ)所述核苷酸序列具有至少90%序列一致性的核苷酸序列。
本发明还提供了表达载体,包括所述核酸分子。
此外,本发明还提供了抗原,包括所述尼帕病毒受体结合糖蛋白。
本发明还提供了表达所述尼帕病毒受体结合糖蛋白的方法,将所述表达载体转染哺乳动物细胞,培养,收集培养基上清,纯化。
更重要的是,本发明还提供了所述尼帕病毒受体结合糖蛋白,所述的核酸分子,所述的表达载体或所述的抗原,在制备如下任一项或多项中的应用;
(I)、制备尼帕病毒的抗体;
(II)、制备和/或评价预防尼帕病毒的疫苗;
(III)、制备治疗尼帕病毒导致疾病的药物;
(IV)、制备尼帕病毒的检测试剂或检测试剂盒;
(V)、研究尼帕病毒的蛋白结构;和/或
(VI)、研究尼帕病毒的入侵机制。
在本发明的一些具体实施方案中,所述尼帕病毒导致疾病包括侵害中枢神经***的急性损伤和/或呼吸***的急性损伤。
更重要的是,本发明还提供了尼帕病毒抗体、预防尼帕病毒的疫苗、治疗尼帕病毒导致疾病的药物和/或尼帕病毒的检测试剂或检测试剂盒,包括所述尼帕病毒受体结合糖蛋白,所述核酸分子,所述表达载体或所述抗原以及可接受的辅料、助剂或载体。
参照GenBank中尼帕病毒受体结合蛋白(G蛋白)编码序列,使用DNAStar软件对各基因型尼帕病毒G蛋白进行核苷酸、氨基酸同源性分析,并绘制进化树,选择代表性毒株(NCBI登录号:AF212302.2),截取合成G蛋白头部结构域(sG)的核苷酸序列,连接至pcDNA3.1(+)真核表达载体上,构建为重组质粒pcDNA-sG,将测序正确的重组质粒在Expi293F细胞中进行表达,利用蛋白免疫印迹(Westernblot)进行鉴定。通过Strep-TactinXT亲和层析纯化目的蛋白,薄层扫描分析蛋白纯度。将获得的sG免疫小鼠以制备抗sG蛋白的多克隆抗体血清,并通过间接ELISA鉴定结合活性。结果表明:尼帕病毒G蛋白在Expi293F细胞中获得可溶性表达,纯化得到的目的蛋白大小正确,纯度大于99%;制备的抗血清能特异性结合目的蛋白。上述研究为尼帕病毒诊断与疫苗研究奠定了基础。
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。
图1(A)示进化树;图1(B)示G蛋白同源性分析结果;
图2(A)示NiV完整G蛋白结构示意图;图2(B)示可溶性G蛋白结构示意图;
图3示pcDNA-sG真核表达质粒图谱;
图4示重组质粒pcDNA-sG酶切鉴定结果;
图5(A)未纯化蛋白SDS-PAGE分析结果;图5(B)示Westernblot鉴定结果;
图6(A)示SDS-PAGE分析结果;图6(B)示E2薄层色谱分析法测蛋白纯度;
图7(A)示SDS-PAGE分析结果;图7(B)示未纯化蛋白样品sG-MS薄层扫描结果;图7(C)示纯化后蛋白样品E2薄层扫描结果;
图8示OD值读数及特异性抗体效价。
具体实施方式
本发明公开了尼帕病毒受体结合糖蛋白及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
尼帕病毒(NiV)作为一种新出现的***共患副粘病毒,能引起人类严重且致命的呼吸道和神经***疾病。这种病毒是在马来西亚和新加坡的养猪户暴发脑炎后首次发现,随后孟加拉国或印度几乎每年都存在尼怕病毒感染病例报告。由于NiV的高致病性、潜在的大流行性以及缺乏许可的疫苗或治疗方法,需要研究和开发高度敏感和特定的诊断工具以及抗病毒药物和疫苗,以帮助预防和控制未来的疫情。就疫苗而言,几乎所有中和抗体都针对的是主要的病毒包膜糖蛋白,在另外两种副黏病毒科成员,流行性腮腺炎和麻疹病毒中,中和抗体是疫苗诱导的主要保护机制,受体结合蛋白(G蛋白)通常是主要的靶标。
本研究以哺乳动物细胞真核表达***表达NiV的受体结合蛋白(G蛋白),有学者使用大肠杆菌原核表达***并选择pET-28a(+)作为表达载体表达尼帕病毒N蛋白,相较于原核表达***,虽然表达蛋白量接近,但哺乳动物细胞可以更准确地完成许多翻译后加工功能,例如糖基化,磷酸化和蛋白水解,因此表达的产物在分子结构,物理和化学性质以及生物学功能方面更接近天然蛋白质。Bossart等采用构建的带有尼帕病毒sG蛋白表达序列以及SGS-tag标签的重组痘苗病毒感染HeLa细胞制备NiV可溶性G蛋白,并通过S蛋白纯化***纯化目的蛋白,而本次实验选用的293F细胞悬浮培养***,对比于HeLa细胞此类贴壁培养细胞,其培养体系更大。本实验在G蛋白上引入了组织纤溶酶原激活物信号肽,使其作为分泌型蛋白产出至培养基内相较于前人的研究通过破碎细胞得到目的蛋白,分泌表达可以使目的蛋白减少胞内蛋白酶的降解,同时有利于对目的蛋白的纯化。结果证明可以有效的减小蛋白纯化的难度。另外,加入了可切除信号肽的TEV蛋白酶酶切位点,能够对标签进行切除,保证了其在后期作为亚单位疫苗使用的安全性。设计截短的重组G蛋白既能实现可溶性表达又保留了良好的免疫原性,并通过免疫小鼠制得具有良好特异性抗体效价的血清,为今后疫苗评价,病毒入侵机制研究奠定了基础。
细胞、菌株、质粒以及实验动物
Expi293F细胞购自Invitrogen,感受态细胞Trans1-T1购自全氏金生物科技、真核表达载体pcDNA-3.1(+)由本实验室保存,6-8周龄雌性BALB/c小鼠购自斯贝福(北京)生物技术有限公司。
主要试剂
抗Strep(HRP)抗体购自MerckMillipore公司、Goat Anti-Mouse IgG H&L(HRP)购自碧云天生物技术有限公司公司,DNA Maker DL5000购自大连TaKaRa公司,T4 DNA Ligase购自NEB公司,胶回收试剂盒购自BioFluX公司,限制性内切酶、Expi293细胞培养基及转染试剂购自Thermo Fisher Scientific公司,质粒提取试剂盒购自Corning公司,Strep-Tactin XT亲和层析柱购自IBA公司,ELISA终止液、TMB单组分显色液均购自南京索莱宝有限公司。
SEQ ID No.1
RPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVLTVNPLVVNWRNNTVISRPGQSQCPRFNTCPEICWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT
SEQ ID No.2
CGGCCTCAGACAGAAGGCGTGTCGAACCTGGTGGGCCTGCCTAACAACATTTGTCTGCAGAAGACCTCTAACCAGATCCTGAAACCTAAACTGATCAGCTACACCCTGCCTGTGGTGGGCCAAAGCGGAACATGCATCACAGACCCCCTCCTGGCCATGGACGAGGGCTATTTCGCCTACTCTCATCTGGAAAGAATCGGCTCTTGCAGCAGAGGCGTGTCCAAGCAGAGAATCATTGGAGTGGGAGAAGTGCTGGATCGGGGAGATGAGGTGCCGTCTCTGTTCATGACCAACGTGTGGACCCCTCCAAATCCAAACACCGTCTACCACTGCAGCGCCGTGTACAACAATGAATTTTACTACGTGCTGTGCGCCGTGTCCACCGTCGGAGATCCTATCCTCAACAGCACCTACTGGAGCGGCAGCCTGATGATGACAAGACTGGCTGTGAAGCCCAAGAGCAACGGCGGAGGATATAATCAACACCAGCTGGCCCTGCGGTCCATCGAGAAGGGCAGATACGATAAGGTTATGCCCTACGGCCCTAGCGGCATCAAGCAGGGCGATACACTGTACTTCCCCGCCGTGGGCTTCCTGGTCCGGACCGAGTTCAAGTACAACGACAGCAACTGCCCCATTACAAAGTGCCAGTACAGCAAACCCGAGAATTGTAGACTGAGCATGGGCATCCGGCCCAACAGCCACTACATCCTGAGAAGCGGCCTGCTGAAGTACAACCTGTCTGACGGCGAGAACCCTAAGGTGGTGTTCATCGAGATCAGCGATCAGCGGCTGTCTATCGGCTCCCCTAGCAAGATCTACGACTCCCTGGGCCAACCTGTGTTCTACCAGGCCAGCTTCAGCTGGGACACAATGATCAAGTTCGGCGACGTGCTTACAGTTAATCCCCTGGTCGTGAACTGGCGGAACAACACCGTGATCAGCAGACCTGGCCAGAGCCAGTGCCCCAGATTCAACACATGTCCTGAGATCTGCTGGGAGGGCGTGTACAACGACGCTTTCCTGATCGACAGAATCAATTGGATCAGCGCCGGCGTGTTTCTGGATAGCAACCAGACCGCCGAGAACCCAGTGTTTACCGTGTTCAAGGACAACGAAATCCTGTACAGAGCCCAGCTGGCCAGCGAGGACACCAATGCGCAGAAAACCATCACCAACTGCTTCCTGCTGAAGAACAAGATCTGGTGCATCAGCCTGGTGGAAATCTATGATACCGGCGACAACGTGATCAGACCTAAGCTGTTTGCCGTTAAGATCCCTGAACAGTGTACA
本发明提供的尼帕病毒受体结合糖蛋白及其应用中,所用原料及试剂均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1:尼帕病毒G蛋白序列分析、设计以及合成
根据NCBI上尼帕病毒的28个全基因组序列,将G蛋白的基因序列进行对比,绘制进化树,并分析G蛋白的氨基酸同源性。通过线上疏水性分析工具、抗原决定簇预测工具(http://www.detaibio.com/tools/)进行分析,得到尼帕病毒代表毒株(NCBI登录号:AF212302.2)的G蛋白序列,删除细胞质尾部区、跨膜疏水区以及部分茎区,仅保留胞外球形头部区(命名为sG)。N端添加人组织纤溶酶原激活物信号肽(tPA)序列、Strep标签,两端分别添加酶切位点HindⅢ和Bam HⅠ,序列设计完成后由上海生工有限公司进行密码子优化并合成,得到含有目的基因质粒(pUC57-NiV-sG)的甘油菌。
实施例2:重组质粒的构建
将合成的含有目的基因质粒(pUC57-NiV-sG)的10μL甘油菌接种于5mL带有氨苄霉素抗性的液体LB培养基,37℃震荡培养12h,进行质粒的小量提取制备,Hind III和Bam HI双酶切后连接至真核表达载体pcDNA3.1(+),得到真核表达质粒pcDNA-sG,将重组质粒采用热激法转化感受态细胞Trans1-T1,经氨苄抗性筛选,挑取阳性菌落,提取质粒,双酶切鉴定,将鉴定正确的质粒送至上海生工生物工程有限公司测序,序列比对确认无误后,大量制备与纯化,测定质粒浓度,用于细胞转染。
实施例3:细胞转染
转染前一天,Expi293F细胞在8%CO2、37℃、125r/min条件下悬浮培养,将60mL体系的293F细胞培养物测定存活率以及活细胞密度,活细胞生长至4.2×106个/mL,存活率为97%,将细胞培养物稀释为2.1×106个/mL,转染当天,细胞密度为3×106个/mL,将60μg真核表达质粒pcDNA-sG与3mL Opti-MEMTMI Reduced Serum Medium混匀,160μLExpiFectamineTM293Reagent与2.8mL Opti-MEMTMI Reduced Serum Medium混匀,室温孵育5min后将二者混匀继续室温下孵育20min。然后将溶液缓慢转移到的细胞培养物摇瓶中,在加入过程中轻轻旋转摇瓶。
实施例4:目的蛋白Western blot检测
转染后12h,加入300μL转染增强剂1和3mL的转染增强剂2,继续培养96h,将培养物以800g离心10min,分别收集培养基上清与细胞沉淀,培养基上清命名为sG-MS,细胞沉淀用IP裂解液重悬后冰上裂解10min,使用超声细胞破碎机破碎10min,离心收集上清命名为sG-DS。蛋白样品进行12%PAGE凝胶电泳,将分离的蛋白电转移至NC(0.45μm)膜上,5%脱脂乳封闭1h后,鼠源抗Strep(HRP)一抗室温孵育2h,TBST洗涤3次每次10min,再与Goat Anti-Mouse IgG H&L(HRP)二抗室温孵育1h,TBST漂洗3次每次10min,用显影仪显影。
实施例5:目的蛋白的纯化
将sG-MS蛋白样品与Strep-Tactin XT亲和层析柱置于垂直混合器混合30min后使样品流穿层析柱,收集流穿液(FT),再用Wash Buffer洗涤5次,分别收集5次的洗涤液命名为W1-5,然后用BufferBXT洗脱目的蛋白3次,第一次3mL,第二次8mL,第三次4mL,分别命名为1号管(E1)、2号管(E2)、3号管(E3)。
实施例6:纯化后目的蛋白SDS-PAGE鉴定以及纯度检测
分别将E1、E2、E3、FT、W1-5进行12%SDS-PAGE凝胶电泳,将凝胶用考马斯亮蓝染色后,脱色12h后进行薄层色谱分析法测蛋白纯度。为进一步确定纯化效率,将E2与未纯化蛋白样品sG-MS进行12%SDS-PAGE凝胶电泳,薄层色谱分析蛋白纯度,BCA法对洗脱峰蛋白(E2)样品进行定量。
实施例7:小鼠免疫
将纯化得到的sG蛋白与弗氏佐剂均匀混合,每只小鼠腹腔注射50μg,阴性对照小鼠腹腔注射100μL PBS,在第0、14和28d分别免疫,共免疫3次;第42d时小鼠眼眶后静脉丛采血,37℃2h后,3500r/min转离心10min分离血清。
实施例8:间接ELISA法检测特异性抗体
实验组与阴性对照组每孔包被5μg纯化后的sG蛋白,空白对照组不包被蛋白,4℃孵育过夜,PBST震荡洗涤3次,每次2min,将实验组以及阴性对照小鼠血清梯度稀释为1:100、1:200、1:400、1:800、1:1600、1:3200、1:6400、1:12800,37℃孵育1h,PBST震荡洗涤3次,每次2min,使用1:5000稀释的Goat Anti-Mouse IgG H&L(HRP)二抗37℃孵育1h,PBST震荡洗涤6次,每次2min,避光加入TMB显色液(100μL/孔),37℃/RT避光孵育15min后加入ELISA终止液,使用多功能酶标仪读取D450nm/D630nm值。
效果例1:尼帕病毒G蛋白遗传进化及氨基酸同源性分析
选择代表性毒株(NCBI登录号:AF212302.2),并下载NCBI上尼帕病毒28个基因型全序列,单独截取G蛋白序列,利用DNAStar软件绘制进化树(图1A),将所选择毒株的G蛋白序列与其他基因型序列进行了氨基酸同源性分析,结果显示,尼帕病毒G蛋白在各基因型中高度保守,且所选择毒株的G蛋白与其他毒株氨基酸同源性介于95%-100%(图1B)。
效果例2:目的基因的设计与合成
以尼帕病毒代表毒株(NCBI ID:AF212302.2)G蛋白为基础,利用表位预测工具进行预测,发现在氨基酸残基183-185aa、417aa、447aa、570aa位置构成了不连续的主要表位,根据预测结果并分析G蛋白结构,拟表达的目的蛋白保留氨基酸残基172-602aa位置,即球形头部结构域(图2A),删除细胞质尾部区、跨膜疏水区以及部分外茎区,命名为sG。同时,N段添加人组织纤溶酶原激活物信号肽(tPA)序列、Strep标签以及可切除标签的TEV蛋白酶酶切序列,两端分别添加酶切位点HindⅢ和Bam HⅠ(图2B)。
效果例3:含有sG蛋白真核表达质粒的构建与鉴定
Hind III/Bam HI双酶切含有目的基因的质粒pUC-NiV-sG与真核表达载体pcDNA3.1(+),将目的基因连接到表达载体中得到重组质粒pCDNA-sG(图3),进行酶切鉴定,结果如图4所示,可见5500bp左右的载体片段和1600bp左右的目的片段(sG),与理伦值相符。对目的基因进行测序,结果与所设计的原序列一致,无碱基突变或缺失,表明成功构建了含有sG蛋白的真核表达质粒。
效果例4:sG蛋白的表达、鉴定、纯化以及定量
sG蛋白的相对分子质量约为60kDa,将培养基上清蛋白样品sG-MS以及细胞破碎产物上清蛋白样品sG-DS分别进行SDS-PAGE和Western印迹分析(图5)。结果显示表达蛋白与预期相符,大小正确且分泌在细胞培养基上清中。分别将纯化后的蛋白1号管(E1)、2号管(E2)、3号管(E3)以及流穿液(FT)、W1-5进行12%SDS-PAGE凝胶电泳,将凝胶用考马斯亮蓝染色后,脱色12h后,对E2进行薄层色谱分析法测蛋白纯度(图6),纯化后蛋白大小正确,约为60KDa,流穿液中为未吸附在层析柱而滤掉的杂蛋白,且洗涤液中未见条带,蛋白纯度检测结果显示纯度为99.9%。为直观评价纯化效率,将纯化后样品(E2)与未纯化蛋白样品(sG-MS)进行12%SDS-PAGE凝胶电泳,同样进行薄层色谱分析法测蛋白纯度(图7),结果显示未纯化蛋白纯度为80.19%,纯化后蛋白纯度为99.51%,纯化前后蛋白纯度提升接近10%,将E2进行BCA定量,蛋白浓度约为1.13μg/μL。证明成功制备了尼帕病毒可溶性受体结合蛋白(sG)。
效果例5:尼帕病毒sG蛋白特异性血清制备与验证
通过免疫小鼠进行血清制备,从首免后每周采血,最终通过间接ELISA确定初免后42d血清OD值读数最高,利用间接ELISA测定小鼠血清中抗G蛋白的特异性抗体效价(图8),读取OD450nm/OD630nm数值,绘制折线图,结果显示小鼠血清中特异性抗G蛋白抗体的效价在1:12800以上,表明重组表达的可溶性G蛋白有效地激发了小鼠体液免疫反应,所制备的血清结合活性良好,可用于后续研究。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 军事科学院军事医学研究院军事兽医研究所
<120> 尼帕病毒受体结合糖蛋白及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 431
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Pro Gln Thr Glu Gly Val Ser Asn Leu Val Gly Leu Pro Asn Asn
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Ile Cys Leu Gln Lys Thr Ser Asn Gln Ile Leu Lys Pro Lys Leu Ile
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Ser Tyr Thr Leu Pro Val Val Gly Gln Ser Gly Thr Cys Ile Thr Asp
35 40 45
Pro Leu Leu Ala Met Asp Glu Gly Tyr Phe Ala Tyr Ser His Leu Glu
50 55 60
Arg Ile Gly Ser Cys Ser Arg Gly Val Ser Lys Gln Arg Ile Ile Gly
65 70 75 80
Val Gly Glu Val Leu Asp Arg Gly Asp Glu Val Pro Ser Leu Phe Met
85 90 95
Thr Asn Val Trp Thr Pro Pro Asn Pro Asn Thr Val Tyr His Cys Ser
100 105 110
Ala Val Tyr Asn Asn Glu Phe Tyr Tyr Val Leu Cys Ala Val Ser Thr
115 120 125
Val Gly Asp Pro Ile Leu Asn Ser Thr Tyr Trp Ser Gly Ser Leu Met
130 135 140
Met Thr Arg Leu Ala Val Lys Pro Lys Ser Asn Gly Gly Gly Tyr Asn
145 150 155 160
Gln His Gln Leu Ala Leu Arg Ser Ile Glu Lys Gly Arg Tyr Asp Lys
165 170 175
Val Met Pro Tyr Gly Pro Ser Gly Ile Lys Gln Gly Asp Thr Leu Tyr
180 185 190
Phe Pro Ala Val Gly Phe Leu Val Arg Thr Glu Phe Lys Tyr Asn Asp
195 200 205
Ser Asn Cys Pro Ile Thr Lys Cys Gln Tyr Ser Lys Pro Glu Asn Cys
210 215 220
Arg Leu Ser Met Gly Ile Arg Pro Asn Ser His Tyr Ile Leu Arg Ser
225 230 235 240
Gly Leu Leu Lys Tyr Asn Leu Ser Asp Gly Glu Asn Pro Lys Val Val
245 250 255
Phe Ile Glu Ile Ser Asp Gln Arg Leu Ser Ile Gly Ser Pro Ser Lys
260 265 270
Ile Tyr Asp Ser Leu Gly Gln Pro Val Phe Tyr Gln Ala Ser Phe Ser
275 280 285
Trp Asp Thr Met Ile Lys Phe Gly Asp Val Leu Thr Val Asn Pro Leu
290 295 300
Val Val Asn Trp Arg Asn Asn Thr Val Ile Ser Arg Pro Gly Gln Ser
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Gln Cys Pro Arg Phe Asn Thr Cys Pro Glu Ile Cys Trp Glu Gly Val
325 330 335
Tyr Asn Asp Ala Phe Leu Ile Asp Arg Ile Asn Trp Ile Ser Ala Gly
340 345 350
Val Phe Leu Asp Ser Asn Gln Thr Ala Glu Asn Pro Val Phe Thr Val
355 360 365
Phe Lys Asp Asn Glu Ile Leu Tyr Arg Ala Gln Leu Ala Ser Glu Asp
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Thr Asn Ala Gln Lys Thr Ile Thr Asn Cys Phe Leu Leu Lys Asn Lys
385 390 395 400
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Ile Arg Pro Lys Leu Phe Ala Val Lys Ile Pro Glu Gln Cys Thr
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<210> 2
<211> 1293
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggcctcaga cagaaggcgt gtcgaacctg gtgggcctgc ctaacaacat ttgtctgcag 60
aagacctcta accagatcct gaaacctaaa ctgatcagct acaccctgcc tgtggtgggc 120
caaagcggaa catgcatcac agaccccctc ctggccatgg acgagggcta tttcgcctac 180
tctcatctgg aaagaatcgg ctcttgcagc agaggcgtgt ccaagcagag aatcattgga 240
gtgggagaag tgctggatcg gggagatgag gtgccgtctc tgttcatgac caacgtgtgg 300
acccctccaa atccaaacac cgtctaccac tgcagcgccg tgtacaacaa tgaattttac 360
tacgtgctgt gcgccgtgtc caccgtcgga gatcctatcc tcaacagcac ctactggagc 420
ggcagcctga tgatgacaag actggctgtg aagcccaaga gcaacggcgg aggatataat 480
caacaccagc tggccctgcg gtccatcgag aagggcagat acgataaggt tatgccctac 540
ggccctagcg gcatcaagca gggcgataca ctgtacttcc ccgccgtggg cttcctggtc 600
cggaccgagt tcaagtacaa cgacagcaac tgccccatta caaagtgcca gtacagcaaa 660
cccgagaatt gtagactgag catgggcatc cggcccaaca gccactacat cctgagaagc 720
ggcctgctga agtacaacct gtctgacggc gagaacccta aggtggtgtt catcgagatc 780
agcgatcagc ggctgtctat cggctcccct agcaagatct acgactccct gggccaacct 840
gtgttctacc aggccagctt cagctgggac acaatgatca agttcggcga cgtgcttaca 900
gttaatcccc tggtcgtgaa ctggcggaac aacaccgtga tcagcagacc tggccagagc 960
cagtgcccca gattcaacac atgtcctgag atctgctggg agggcgtgta caacgacgct 1020
ttcctgatcg acagaatcaa ttggatcagc gccggcgtgt ttctggatag caaccagacc 1080
gccgagaacc cagtgtttac cgtgttcaag gacaacgaaa tcctgtacag agcccagctg 1140
gccagcgagg acaccaatgc gcagaaaacc atcaccaact gcttcctgct gaagaacaag 1200
atctggtgca tcagcctggt ggaaatctat gataccggcg acaacgtgat cagacctaag 1260
ctgtttgccg ttaagatccc tgaacagtgt aca 1293
Claims (10)
1.尼帕病毒受体结合糖蛋白,其特征在于,以尼帕病毒G蛋白为基础,保留氨基酸残基172-602aa位置,删除细胞质尾部区、跨膜疏水区以及氨基酸残基71-171位置的外茎区;N段添加人组织纤溶酶原激活物信号肽序列、Strep标签以及可切除标签的TEV蛋白酶酶切序列,两端分别添加酶切位点HindⅢ和Bam H Ⅰ。
2.如权利要求1所述的尼帕病毒受体结合糖蛋白,其特征在于,其具有:
(I)、如SEQ ID No.1所示的氨基酸序列;或
(II)、如(I)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列,且与(I)所述的氨基酸序列功能相同的氨基酸序列;或
(III)、与如(I)或(II)所述的氨基酸序列具有90%以上同一性的氨基酸序列;
所述取代、缺失或添加一个或多个氨基酸中的4个或5个。
3.编码如权利要求1或2所述尼帕病毒受体结合糖蛋白的核酸分子。
4.如权利要求3所述的核酸分子,其特征在于,其具有:
(Ⅰ)、如SEQ ID No.2所示的核苷酸序列;或
(Ⅱ)、如SEQ ID No.2所示的核苷酸序列的互补核苷酸序列;或
(Ⅲ)、与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质,但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的核苷酸序列;或
(Ⅳ)、与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列经取代、缺失或添加一个或两个核苷酸序列获得的核苷酸序列,且与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(V)、与(Ⅰ)、(Ⅱ)、(Ⅲ)或(Ⅳ)所述核苷酸序列具有至少90%序列一致性的核苷酸序列。
5.表达载体,其特征在于,包括如权利要求3或4所述的核酸分子。
6.抗原,其特征在于,包括如权利要求1或2所述尼帕病毒受体结合糖蛋白。
7.表达如权利要求1或2所述尼帕病毒受体结合糖蛋白的方法,其特征在于,将如权利要求5所述的表达载体转染哺乳动物细胞,培养,收集培养基上清,纯化。
8.如权利要求1或2所述尼帕病毒受体结合糖蛋白,如权利要求3或4所述的核酸分子,如权利要求5所述的表达载体或如权利要求6所述的抗原,在制备如下任一项或多项中的应用;
(I)、制备尼帕病毒的抗体;
(II)、制备和/或评价预防尼帕病毒的疫苗;
(III)、制备治疗尼帕病毒导致疾病的药物;
(IV)、制备尼帕病毒的检测试剂或检测试剂盒;
(V)、研究尼帕病毒的蛋白结构;和/或
(VI)、研究尼帕病毒的入侵机制。
9.如权利要求8所述的应用,其特征在于,所述尼帕病毒导致疾病包括侵害中枢神经***的急性损伤和/或呼吸***的急性损伤。
10.尼帕病毒抗体、预防尼帕病毒的疫苗、治疗尼帕病毒导致疾病的药物和/或尼帕病毒的检测试剂或检测试剂盒,其特征在于,包括如权利要求1或2所述尼帕病毒受体结合糖蛋白,如权利要求3或4所述的核酸分子,如权利要求5所述的表达载体或如权利要求6所述的抗原以及可接受的辅料、助剂或载体。
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