CN106632620A - 登革病毒重组抗原及其制备方法和应用 - Google Patents
登革病毒重组抗原及其制备方法和应用 Download PDFInfo
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- CN106632620A CN106632620A CN201611074460.4A CN201611074460A CN106632620A CN 106632620 A CN106632620 A CN 106632620A CN 201611074460 A CN201611074460 A CN 201611074460A CN 106632620 A CN106632620 A CN 106632620A
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Abstract
本发明公开了一种登革病毒重组抗原及其制备方法和应用,登革病毒重组抗原包括:(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽;(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98%同源性的多肽;或(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽,其中一个或多个氨基酸被缺失、替代或增加得到的多肽。经过试验验证,这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本,因此,这种登革病毒重组抗原可以用于登革热检测。
Description
技术领域
本发明涉及病毒检测领域,特别是涉及一种登革病毒重组抗原及其制备方法和应用。
背景技术
登革热是由DEN-1、DEN-2、DEN-3和DEN-4四种血清型登革病毒引起的急性蚊媒传染病,各血清型均可引起登革热和致死率很高的登革出血热以及登革休克综合症。登革热广泛流行于全球热带和亚热带的非洲、美洲、东南亚和西太平洋区的100多个国家和地区,目前我国主要流行于广东、广西、海南和台湾地区,其主要传播媒介是埃及伊蚊和白蚊伊蚊。世界卫生组织估计全球每年登革热发病人数约5千万到1亿,并导致25到50万登革出血热病例和2.4万人死亡。登革热爆发疫情来势急,扩散快,登革热疑似病例的尽快确诊直接关系到及早发现疫情。
登革病毒是登革热的病原体,黄病毒科黄病毒属主要成员,是有包膜的单股正链RNA分子,基因组长约11kb。其基因组的5’端有帽子结构,3’端为CUOH,无多聚A结构。cDNA序列分析表明,登革病毒RNA的5’端和3’端各有一段非编码区,中间仅有一个开放阅读框(ORF),基因组约96%的核苷酸序列在此框架内。该ORF序列编码病毒的3种结构蛋白(核心蛋白c、膜结合蛋白前体PrM和包膜蛋白E)和7种非结构蛋白(Ns)。整个基因组的编码顺序为:5’-C-PrM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3’。经免疫学研究发现,只有C、PrM、E、NS1和NS3既具有反应原性,又具有免疫原性。
登革病毒E蛋白是登革病毒最大的结构蛋白和主要的包膜糖蛋白,也是登革病毒的主要毒力蛋白,而且还带有型特异、属特异抗原决定簇。它也是登革病毒吸附靶细胞、致病及诱导免疫保护的关键性蛋白,在登革病毒的致病机理及诱发机体的抗感染免疫中发挥重要的作用,对其结构和功能的分析一直成为国内外登革研究的重点。E蛋白为登革病毒VAP(病毒表面特异性吸附蛋白,virus attachment protein),即宿主细胞表面病毒受体的配体,介导病毒特异性地与靶细胞结合。在中性和微碱性的条件下,成熟病毒体表面的E蛋白是以同源二聚体的形式存在,当E蛋白作为VAP介导病毒颗粒和靶细胞结合,以胞质内吞的途径入胞,吞噬体所处的内环境的pH值有所下降,E蛋白构象重排,由同源二聚体变为三聚体结构。构象的变化引起宿主细胞膜和病毒膜的融合,使得病毒基因组进入胞浆,引起感染。
登革热临床表现复杂多样、传播迅速、发病率高、人群易感染,严重影响了人们的生活质量和身体健康。而目前对登革感染仍缺乏疫苗及特效药物治疗,因此,特异、敏感、快速的诊断方法对登革热的流行和预防控制有重要意义。
发明内容
基于此,有必要提供一种能够用于登革热检测的登革病毒重组抗原及其制备方法和应用。
一种登革病毒重组抗原,包括:
(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽;
(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98%同源性的多肽;或
(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽,其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
一种上述的登革病毒重组抗原的制备方法,包括如下步骤:
步骤一、提供基因表达载体,所述基因表达载体用于表达所述登革病毒重组抗原;
步骤二、将所述基因表达载体转化到原核表达菌中;
步骤三、对转化了所述基因表达载体的所述原核表达菌进行诱导表达,表达完成后收菌裂解,离心后保留第一沉淀;
步骤四、用缓冲液清洗所述第一沉淀后再次离心,保留第二沉淀,使用复溶液将所述第二沉淀再溶解,得到粗产物溶液;以及
步骤五、对所述粗产物溶液过Ni-NTA亲和柱纯化,再进行蛋白复性,得到所述登革病毒重组抗原。
在一个实施例中,所述缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100,所述缓冲液的pH为8;
所述复溶液包括6M的尿素和20mM的Tris,所述复溶液的pH为8。
在一个实施例中,所述对所述粗产物溶液过Ni-NTA亲和柱纯化的操作中,平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素,所述平衡液的pH为8,洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑,所述洗脱液的pH为8。
在一个实施例中,所述进行蛋白复性的操作为:分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行透析复性;
所述第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第一复性缓冲液的pH为8;
所述第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第二复性缓冲液的pH为8;
所述第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第三复性缓冲液的pH为8。
上述的登革病毒重组抗原在制备登革病毒检测试剂领域或制备登革病毒检测设备领域的应用。
一种登革病毒检测试纸,含有上述的登革病毒重组抗原。
在一个实施例中,包括支撑薄片、样品垫、金标垫、硝酸纤维素膜、吸收垫、T1检测线、T2检测线及质控线;
所述样品垫、所述金标垫、所述硝酸纤维素膜和所述吸收垫从所述支撑薄片的一端向另一端依次设置在所述支撑薄片上,所述样品垫与所述金标垫部分重叠,所述金标垫与所述硝酸纤维素膜部分重叠,所述硝酸纤维素膜与所述吸收垫部分重叠;
所述T1检测线、所述T2检测线和所述质控线均设在所述硝酸纤维素膜上,所述T1检测线设在所述硝酸纤维素膜靠近所述金标垫的一端,所述T2检测线设在所述硝酸纤维素膜靠近所述吸收垫的一端,所述质控线位于所述T1检测线和所述T2检测线中间;
所述金标垫上涂覆有胶体金标记的鼠IgG和胶体金标记的所述登革病毒重组抗原,所述T1检测线为抗人IgG抗体,所述T2检测线为抗人IgM抗体,所述质控线为羊抗鼠IgG抗体。
一种登革病毒检测试剂盒,包括上述的登革病毒检测试纸。
在一个实施例中,还包括壳体,所述登革病毒检测试纸设置在所述壳体内,并且所述壳体上设有加样孔和观察窗,所述加样孔与所述样品垫对应设置,所述T1检测线、所述T2检测线和所述质控线与所述观察窗对应设置。
经过试验验证,这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本,因此,这种登革病毒重组抗原可以用于登革热检测。
附图说明
图1为一实施方式的登革病毒检测试剂盒的结构示意图;
图2为如图1所示的登革病毒检测试剂盒的登革病毒检测试纸的正面结构示意图;
图3为如图1所示的登革病毒检测试剂盒的登革病毒检测试纸的剖面结构示意图;
图4为实施例1中得到的第一沉淀的SDS-PAGE电泳图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施的限制。
本发明公开了一种登革病毒重组抗原,包括:
(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽;
(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98%同源性的多肽;或
(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽,其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
经过试验验证,这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本,因此,这种登革病毒重组抗原可以用于登革热检测。
本发明还公开了上述登革病毒重组抗原的制备方法,包括如下步骤:
步骤一、提供基因表达载体,基因表达载体用于表达上述登革病毒重组抗原。
基因表达载体的制备方法如下:首先在Genbank中搜集登革I~IV型病毒株基因的序列,建立登革I~IV型病毒株的基因数据,并且利用计算机软件对其进行分析,确定最具有检测活性的区段,将登革热4个血清型的最具检测活性的区段的基因序列直接串联起来后,进行人工合成,并在其上游引物引入BamHI位点,下游引物引入EcoRI位点且EcoRI位点之前带有6个His氨基酸的编码序列以及终止密码TAA,具体的氨基酸序列见SEQ ID No.1,合成目的片段后,用上下游引物进行PCR扩增,扩增获得的PCR片段经回收(本发明所使用的分子生物学提取和回收试剂盒均购自上海华舜生物工程有限公司)之后,用BamHI和EcoRI酶切(本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司),连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(Novagen公司,货号:69864-3)中,得到的重组质粒pET-24a-DENI~IV即为所需的基因表达载体。
步骤二、将步骤一得到的基因表达载体转化到原核表达菌中。
原核表达菌可以为大肠杆菌。具体的,原核表达菌为BL21(DE3)(购自美国Invitrogen公司)。
步骤三、对步骤二得到的转化了基因表达载体的原核表达菌进行诱导表达,表达完成后收菌裂解,离心后保留第一沉淀。
步骤三具体为:将转化了基因表达载体的原核表达菌置于37℃、200rpm摇床过夜培养至菌体浓度为OD600=2.814,加入IPTG(终浓度为0.5mM)进行诱导,190rpm,37度诱导约4h,收菌进行超声裂解,离心分离并保留第一沉淀。
步骤四、用缓冲液清洗步骤三得到的第一沉淀后再次离心,保留第二沉淀,使用复溶液将第二沉淀再溶解,得到粗产物溶液。
缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100,缓冲液的pH为8。
复溶液包括6M的尿素和20mM的Tris,复溶液的pH为8。
步骤五、对步骤四得到的粗产物溶液过Ni-NTA亲和柱纯化,再进行蛋白复性,得到登革病毒重组抗原。
对粗产物溶液过Ni-NTA亲和柱纯化的操作中,平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素,平衡液的pH为8,洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑,洗脱液的pH为8。
进行蛋白复性的操作为:分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行透析复性。
第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第一复性缓冲液的pH为8。
第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第二复性缓冲液的pH为8。
第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第三复性缓冲液的pH为8。
透析复性后得到的登革病毒重组抗原透析至保存液(20mM Tris-HCl,150mMNaCl,pH 8.0)中,收集透析样品,测定蛋白浓度,置于-20℃备用。
这种登革病毒重组抗原可以应用于制备登革病毒检测试剂领域或制备登革病毒检测设备领域。
接下来,以上述登革病毒重组抗原应用于制备登革病毒检测试剂盒为例进行简单介绍。
如图1~图3所示的一实施方式的登革病毒检测试剂盒,包括登革病毒检测试纸100和壳体200,登革病毒检测试纸100设置在壳体200内。
登革病毒检测试纸中含有上述登革病毒重组抗原。
结合图2和图3,登革病毒检测试纸100包括支撑薄片110、样品垫120、金标垫130、硝酸纤维素膜140、吸收垫150、T1检测线160、T2检测线180及质控线170。
样品垫120、金标垫130、硝酸纤维素膜140、吸收垫150从支撑薄片110的一端向另一端依次设置在支撑薄片110上,样品垫120与金标垫130部分重叠,金标垫130与硝酸纤维素膜140部分重叠,硝酸纤维素膜140与吸收垫150部分重叠。
T1检测线160、T2检测线180及质控线170设在硝酸纤维素膜140上,T1检测线160设在硝酸纤维素膜140靠近金标垫130的一端,T2检测线180设在硝酸纤维素膜140靠近吸收垫150的一端,质控线170位于T1检测线160和T2检测线180中间。
金标垫130上涂覆有胶体金标记的鼠IgG和胶体金标记的上述登革病毒重组抗原,T1检测线160为抗人IgG抗体,T2检测线180为抗人IgM抗体,质控线170为羊抗鼠IgG抗体。
结合图1,壳体200上设有加样孔210和观察窗220。加样孔210与样品垫120对应设置,T1检测线160、T2检测线180及质控线170与观察窗220对应设置。
这种登革病毒检测试剂盒利用捕获法来检测被检材料中的是否含有登革病毒特异性的IgG抗体和IgM抗体。检测时,若样品中含有登革病毒特异性的IgG抗体和IgM抗体,IgG抗体和IgM抗体先和胶体金标记的登革病毒重组抗原结合形成抗原抗体复合物,由于毛细管作用,抗原抗体复合物沿硝酸纤维素膜140向前泳动,到达T1检测线160和T2检测线180时,抗原抗体复合物会与相应的IgG抗体和IgM抗体结合,从而富集在T1检测线160和T2检测线180上,形成红色沉淀线。胶体金标记的鼠IgG则通过T1检测线160和T2检测线180,被羊抗鼠IgG抗体捕获,富集在质控线170上,形成红色沉淀线。当T1检测线160和T2检测线180与质控线170上同时有红色沉淀线时判为阳性结果。若样品中不含有登革病毒特异性的IgG抗体和IgM抗体,胶体金标记的登革病毒重组抗原不会与T1检测线160和T2检测线180上的抗体结合,胶体金标记的登革病毒重组抗原则通过T1检测线160和T2被羊抗鼠IgG抗体捕获,并富集在质控线170上形成红色沉淀线,此时判为阴性结果。
此外,在其他实施方式中,登革病毒检测试剂盒的结构不限于上文描述。上述登革病毒重组抗原除应用在上述检测试剂盒外,还可以用于其他的登革病毒检测试剂盒或设备中。本领域技术人员可以理解,将本实施方式的登革病毒重组抗原直接或间接结合其他信号基团(如磁性微球、辣根过氧化酶等),或将本实施方式的登革病毒重组抗原作为包被抗原(例如ELISA),则可用于其他形式的登革病毒检测试剂或设备。故本实施方式所制备得到的登革病毒重组抗原可广泛适用于制备登革病毒检测试剂或设备。
特别需要指出的是,上述登革病毒检测试纸100可以单独使用。
通过使用上述登革病毒重组抗原,大大简化了登革病毒检测试剂盒的制备工艺,且将该登革病毒重组抗原应用于登革病毒检测试剂盒中,其表现了比国际同类产品更高的灵敏度和更强的特异性。
以下为具体实施例。
具体实施例中采用药物和仪器均为本领域常规选择。
实施例1、登革病毒重组抗原的制备
1、登革病毒重组抗原表达质粒的构建
首先在Genbank中搜集登革I~IV型病毒株基因的序列,建立登革I~IV型病毒株的基因数据,并且利用计算机软件对其进行分析,确定最具有检测活性的区段,将登革热4个血清型的最具检测活性的区段的基因序列直接串联起来后,进行人工合成,并在其上游引物引入BamHI位点,下游引物引入EcoRI位点且EcoRI位点之前带有6个His氨基酸的编码序列以及终止密码TAA,具体的氨基酸序列见SEQ ID No.1,合成目的片段后,用上下游引物进行PCR扩增,扩增获得的PCR片段经回收(本发明所使用的分子生物学提取和回收试剂盒均购自上海华舜生物工程有限公司)之后,用BamHI和EcoRI酶切(本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司),连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(Novagen公司,货号:69864-3)中,得到重组质粒pET-24a-DENI~IV,即本发明的标记抗原的重组质粒,表达的蛋白命名为DEN040。将上述构建好的重组质粒pET-24a-DENI~IV转化至宿主表达菌BL21(DE3)(购自美国Invitrogen公司),得到重组质粒的表达菌株,备用。
2、登革病毒重组抗原的原核表达
缓冲液的配制:20mM Tris-HCl,1mM EDTA,50mM NaCl,0.01wt%Triton X-100,pH8.0。
将上述宿主表达菌置于37度200rpm摇床过夜培养至菌体浓度为OD600=2.814,加入IPTG(终浓度为0.5mM)进行诱导,190rpm,37度诱导约4h,收菌进行超声裂解,离心分离上清和第一沉淀。
第一沉淀经SDS-PAGE电泳,得到图4。由图4可以看出,大部分目的蛋白为包涵体表达,保留沉淀进行后续处理。
沉淀用缓冲液洗杂3次,12000rpm×15min离心,去上清保留第二沉淀,第二沉淀用复溶液(6M尿素,20mM Tris,pH8.0)溶解,超声2min,4度环境放置30min后,再超声2min,13500rpm×30min,取上清,待进行Ni亲和。
3、登革病毒重组抗原的纯化和复性
平衡液的配制:20mM Tris-HCl,200mMNaCl,6M尿素,pH8.0。
洗脱液的配制:20mM Tris-HCl,200mMNaCl,6M尿素,再加入250mM咪唑,pH8.0。
配制好平衡液和洗脱液以后,利用ATKA purifier层析仪(购自GE医疗集团),先用平衡液清洗平衡Ni-NTA亲和柱(Qiagen公司,货号30210),Ni-NTA柱平衡好之后,将待上柱样品上柱结合,结合完之后,再用10倍柱体积的平衡液清洗柱子,然后再用洗脱液进行逐步洗脱,收集洗脱峰,测定蛋白浓度,待复性。
将上述Ni-NTA亲和纯化得到的目的蛋白用平衡液稀释浓度为1mg/mL,再分别用含有4M、2M、1M尿素的复性缓冲液(20mM Tris-HCl,150mM NaCl,pH 8.0)于4度进行透析复性,最终将目的蛋白透析至缓冲液(20mM Tris-HCl,150mM NaCl,pH 8.0)中,收集透析样品,测定蛋白浓度,置于-20度备用。
实施例2、登革病毒检测试纸的制备
1、硝酸纤维素膜的制备
包被缓冲液的配制:含6%甲醇、0.01M pH7.2的PBS缓冲液为包被缓冲液,0.22μm膜滤过,置4℃备用,有效期一周。1000mL 6%甲醇的0.01M pH 7.2PBS缓冲液配方:NaCl8g、KCl 0.2g、Na2HPO4·12H2O 2.9g、KH2PO4 0.2g、甲醇60mL,双蒸去离子水定容至1000mL。
硝酸纤维素膜的制备:用包被缓冲液将抗人IgG抗体(菲鹏生物股份有限公司生产,货号BA-PAB-MU0003)稀释到1~2mg/mL,调整机器,划线为T1检测线,即为登革病毒IgG抗体检测线,T1检测线靠近金标垫端,距金标垫端约5mm;用包被缓冲液将抗人IgM抗体(菲鹏生物股份有限公司生产,货号BA-PAB-MU0005)稀释到1~2mg/mL,调整机器,划线为T2检测线,即为登革病毒IgM抗体检测线,T2检测线位于T1检测线上面,距T1检测线约3~5mm;用包被缓冲液将羊抗鼠IgG抗体(菲鹏生物股份有限公司生产,货号BA-PAB-MU0001)稀释到1~5mg/mL,调整机器,划线为质控线,质控线靠近吸收垫,距吸收垫约3mm,与T2检测线距离5~8mm,均匀。37℃烘干,封装备用。
2、胶体金标记的鼠IgG和胶体金标记的登革病毒重组抗原的制备。
(1)溶液的配制。
①氯金酸的配制:用双蒸去离子水溶解氯金酸,配成1%溶液,置4℃备用,有效期四个月。1000mL 1%氯金酸溶液配方:10g氯金酸:双蒸去离子水定容至1000mL。
②柠檬酸三钠的配制:用双蒸去离子水溶解柠檬酸钠,配成1%溶液,0.22μm膜滤过,置4度备用,有效期容至1000mL。
③0.1M碳酸钾的配制:用双蒸去离子水配制,0.22μm膜滤过,置4度备用,有效期四个月。1000mL0.1M碳酸钾溶液配方:13.8g碳酸钾;双蒸去离子水定容至1000mL。
④标记洗涤保存液的配制:2%牛血清白蛋白(BSA),0.05%叠氮钠(NaN3),0.01MpH7.2 PBS溶液,0.22μ膜滤过,置4℃备用,有效期四个月。1000mL标记洗涤保存液配方:20gBSA,0.5g NaN3、0.01M pH7.2 PBS溶液定容至1000mL。
(2)胶体金的制备。
用双蒸去离子水将1%氯金酸稀释成0.01%,置电炉煮沸,按每100mL 0.01%氯金酸加入2mL 1%柠檬酸三钠,继续煮沸,直到液体呈亮红色即停止加热,冷却至室温后补足失水。制备好的胶体金外观应纯净、透亮、无沉淀和漂浮物,有效期一周。
(3)胶体金标记的登革病毒重组抗原的制备。
用0.1M碳酸钾调胶体金的pH值至8.2,按8~10μg抗体/mL胶体金加入实施例1中制备得到的登革病毒重组抗原,磁力搅拌器混匀30min,搅拌下加入BSA至终浓度为1%静置1小时。13000rpm、4℃离心30min,弃上清,沉淀用标记洗涤保存液洗涤两次,用十分之一初始胶体金体积的标记洗涤保存液将沉淀重悬,置4℃备用,有效期一周。
(4)胶体金标记鼠IgG的制备。
用0.1M碳酸钾调胶体金的pH值至8.2,按8~10μg抗体/mL胶体金加入鼠IgG(菲鹏生物股份有限公司,货号BA-PAB-MU0006),磁力搅拌器混匀30min,搅拌下加入BSA至终浓度为1%静置1小时。13000rpm、4℃离心30min,弃上清,沉淀用标记洗涤保存液洗涤两次,用十分之一初始胶体金体积的标记洗涤保存液将沉淀重悬,置4℃备用,有效期一周。
3、金标垫的制备
(1)封闭液的配制。
2%BSA,0.1%TritonX-100、0.05%NaN3,0.01M pH7.2 PBS溶液,0.22μm膜滤过,置4度备用,有效期四个月。1000mL封闭液配方:20g BSA,0.5g NaN3、1mL TritonX-100、0.01M pH7.2 PBS溶液定容至1000mL。
(2)金标垫的制备。
将金标垫浸泡于封闭液中30min后,于37℃烘干。将胶体金标记的登革病毒重组抗原和胶体金标记的鼠IgG按照适当比例混合均匀,然后将混合好的胶体金标记的登革病毒重组抗原和胶体金标记的鼠IgG均匀的铺在金标垫上,每毫升溶液铺20平方厘米,冷冻干燥,封装,置4℃备用。
4、试纸条样品垫的制备
(1)封闭液的配制。
2%BSA,0.1%TrtionX-100、0.05%NaN3,0.01M pH7.2 PBS溶液,0.22μm膜滤过,置4度备用,有效期四个月。1000mL封闭液配方:20g BSA,0.5g NaN3、1mL TrtionX-100、0.01M pH7.2 PBS溶液定容至1000mL。
(2)样品垫的制备。
将样品垫浸泡于封闭液中30min后,于37℃烘干,封装,置4℃备用。
5.登革病毒检测试纸的组装
将吸收垫(购自Millipore公司)、硝酸纤维素膜、金标垫、样品垫设置在不吸水的支撑薄片上,切成3mm宽的小条。每十小条一包,加入干燥剂,真空封装,得到登革病毒检测试纸。
实施例3、登革病毒检测试剂盒
1、快速检测登革病毒抗体IgG/IgM的试剂盒包括:实施例2制作的登革病毒检测试纸、壳体和样品稀释液。
登革病毒检测试纸放置到壳体中即可。
样品稀释液为8%NaCl溶液。配制方法:80gNaCl,加蒸馏水定容至1000mL。
2、胶体金法检测登革病毒抗体IgG/IgM。
(1)直接吸取10μL采集好的人血清或血浆加入到试纸卡加样孔,等待15min后即可观察结果。
(2)结果判定:当试纸条出现肉眼可见的红色质控线,没有出现肉眼可见的红色检测线,结果判为阴性;当试纸条出现肉眼可见的红色质控线,同时也出现肉眼可见的红色检测线,结果判为阳性。检测线颜色越深说明被检测样品的登革病毒抗体IgG或IgM水平越高。当试纸条没有出现肉眼可见的紫红色质控线,不管有没有出现肉眼可见的紫红色检测线,结果都判为检测试纸条失效,应废弃。
实施例4、登革病毒检测试剂盒
以商业化的主流产品SD Dengue IgG/IgM试剂盒和实施例3得到的登革病毒检测试剂盒(简称FP dengue IgG/IgM试剂盒)做对比,同时检测97份登革阳性标本和478份临床阴性标本,这97例阳性标本再通过商品化的登革病毒分型多重PCR试剂盒进行RT-PCR分型检测,其中37份为II型登革病毒感染,23份为I型登革病毒感染、15份为III型登革病毒感染、22份为IV型登革病毒感染。结果显示,FP Dengue IgG/IgM试剂盒能检出上述97份阳性标本中的96份,而SD Dengue IgG/IgM试剂盒检出阳性标本94份,对于478份临床阴性标本,FP试剂盒检出假阳18份,SD试剂盒检出假阳23份,具体见表1。
上述结果说明实施例3得到的登革病毒检测试剂盒在灵敏度和特异性方面均优于SD的试剂盒,完全可以用于登革病毒的快速诊断。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
<110> 菲鹏生物股份有限公司
<120> 登革病毒重组抗原及其制备方法和应用
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 374
<212> PRT
<213> 登革病毒重组抗原
<400> 1
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Claims (10)
1.一种登革病毒重组抗原,其特征在于,包括:
(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽;
(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98%同源性的多肽;或
(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽,其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
2.一种根据权利要求1所述的登革病毒重组抗原的制备方法,其特征在于,包括如下步骤:
步骤一、提供基因表达载体,所述基因表达载体用于表达所述登革病毒重组抗原;
步骤二、将所述基因表达载体转化到原核表达菌中;
步骤三、对转化了所述基因表达载体的所述原核表达菌进行诱导表达,表达完成后收菌裂解,离心后保留第一沉淀;
步骤四、用缓冲液清洗所述第一沉淀后再次离心,保留第二沉淀,使用复溶液将所述第二沉淀再溶解,得到粗产物溶液;以及
步骤五、对所述粗产物溶液过Ni-NTA亲和柱纯化,再进行蛋白复性,得到所述登革病毒重组抗原。
3.根据权利要求2所述的制备方法,其特征在于,所述缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100,所述缓冲液的pH为8;
所述复溶液包括6M的尿素和20mM的Tris,所述复溶液的pH为8。
4.根据权利要求2所述的制备方法,其特征在于,所述对所述粗产物溶液过Ni-NTA亲和柱纯化的操作中,平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素,所述平衡液的pH为8,洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑,所述洗脱液的pH为8。
5.根据权利要求2所述的制备方法,其特征在于,所述进行蛋白复性的操作为:分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行透析复性;
所述第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第一复性缓冲液的pH为8;
所述第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第二复性缓冲液的pH为8;
所述第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl,所述第三复性缓冲液的pH为8。
6.根据权利要求1所述的登革病毒重组抗原在制备登革病毒检测试剂领域或制备登革病毒检测设备领域的应用。
7.一种登革病毒检测试纸,其特征在于,含有根据权利要求1所述的登革病毒重组抗原。
8.根据权利要求7所述的登革病毒检测试纸,其特征在于,包括支撑薄片、样品垫、金标垫、硝酸纤维素膜、吸收垫、T1检测线、T2检测线及质控线;
所述样品垫、所述金标垫、所述硝酸纤维素膜和所述吸收垫从所述支撑薄片的一端向另一端依次设置在所述支撑薄片上,所述样品垫与所述金标垫部分重叠,所述金标垫与所述硝酸纤维素膜部分重叠,所述硝酸纤维素膜与所述吸收垫部分重叠;
所述T1检测线、所述T2检测线和所述质控线均设在所述硝酸纤维素膜上,所述T1检测线设在所述硝酸纤维素膜靠近所述金标垫的一端,所述T2检测线设在所述硝酸纤维素膜靠近所述吸收垫的一端,所述质控线位于所述T1检测线和所述T2检测线中间;
所述金标垫上涂覆有胶体金标记的鼠IgG和胶体金标记的所述登革病毒重组抗原,所述T1检测线为抗人IgG抗体,所述T2检测线为抗人IgM抗体,所述质控线为羊抗鼠IgG抗体。
9.一种登革病毒检测试剂盒,其特征在于,包括如权利要求7或8所述的登革病毒检测试纸。
10.根据权利要求9所述的登革病毒检测试剂盒,其特征在于,还包括壳体,所述登革病毒检测试纸设置在所述壳体内,并且所述壳体上设有加样孔和观察窗,所述加样孔与所述样品垫对应设置,所述T1检测线、所述T2检测线和所述质控线与所述观察窗对应设置。
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