CN114181953A - 一种携带ha标签的乙型脑炎病毒全长感染性克隆的制备方法 - Google Patents
一种携带ha标签的乙型脑炎病毒全长感染性克隆的制备方法 Download PDFInfo
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Abstract
本发明涉及一种携带HA标签的乙型脑炎病毒全长感染性克隆的制备方法及其应用,属于分子生物学领域。为了克服黄病毒NS2A研究过程中缺乏针对NS2A的特异性抗体的技术不足,本发明提供一种携带HA标签的乙型脑炎病毒全长感染性克隆的制备方法,并公开了其应用。本发明通过在NS1与NS2A之间插入HA标签,通过HA标签来指示NS2A,同时为避免影响NS2A荧光信号的观察,对NS2A第30个氨基酸进行点突变,以干扰NS2A的茎环结构,消除病毒复制过程的‑1核糖体移码。本发明所述的携带HA标签的乙型脑炎病毒全长感染性克隆对于黄病毒的研究具有重要的价值。
Description
技术领域
本发明涉及一种携带HA标签的乙型脑炎病毒全长感染性克隆的制备方法,属于分子生物学领域。
背景技术
乙型脑炎(Japanese encephalitis,JE)是由黄病毒科的乙型脑炎病毒(JEvirus,JEV)引起的人和动物共患的一种由蚊虫为媒介传播的病毒性疾病,对人类危害巨大,是人类中枢神经系统最常见的虫媒病之一,对猪可引起繁殖障碍,给养猪业造成巨大的经济损失;猪是JEV的扩增宿主,带毒猪是本病的主要传染源,因此,防制猪患乙脑不但可以减少养猪业的损失,而且也是防制乙脑的重要措施。
乙脑病毒基因为单股正链RNA,长约11kb,有单一开放的阅读框,能编码,翻释,加工成多种结构蛋白和非结构蛋白,其病毒的结构蛋白有3种:囊膜蛋白E,膜蛋白M(由膜前体蛋白PrM加工而来),以及衣壳蛋白C和7种非结构蛋白NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5;其中M蛋白参与病毒囊膜的构成,对维持E蛋白的结构是十分必要的,E蛋白在保护性免疫及防制病毒感染起作重要作用。
目前针对大多数黄病毒,还没有有效的抗病毒药物和疫苗。开发广谱保护性疗法和疫苗成为黄病毒防控领域一个关键而仍未实现的目标。之前的黄病毒治疗靶标和疫苗设计主要聚焦在病毒囊膜表面的E蛋白,如高福院士团队于2016年解析了靶向E蛋白且能交叉保护寨卡病毒的单克隆抗体2A10G6的作用机制1,并从中国寨卡康复病人体内鉴定出高效、特异的靶向寨卡病毒E蛋白的单克隆抗体2。然而,E蛋白诱导的抗体保护范围有限,更为重要的是,登革病毒感染以及疫苗研发过程中,发现E蛋白免疫会诱导产生有交叉反应性但中和能力差的抗体,当机体感染不同类型登革病毒时,这些抗体会产生抗体依赖增强(ADE)效应,反而会导致更加严重的感染。这对开发安全有效的广谱疫苗造成了重大挑战。
在针对黄病毒NS2A研究过程中,遇到的其中一个难题是缺乏针对NS2A的特异性抗体。科研人员通过多种途径制备针对NS2A的特异性抗体,均没有成功。基于此,本发明提供一种携带HA标签的乙型脑炎病毒全长感染性克隆及其制备方法。
发明内容
黄病毒NS2A研究过程中科研人员试图通过多种途径制备针对NS2A的特异性抗体,均没有成功。另外,已知NS2A基因的起始区域存在一个保守七联核苷酸滑动序列(CCCUUUU)和序列后的二级茎环结构,会引起病毒复制过程中发生-1核糖体移码,产生比NS1蛋白大52个氨基酸NS1'蛋白(MELIAN et al.,2010)。我们在NS1-NS2A之间引入HA标签的同时,NS1'蛋白同时也会引入HA标签,这会影响NS2A荧光信号的观察。
为了克服黄病毒NS2A研究过程中缺乏针对NS2A的特异性抗体的技术不足,本发明提供一种携带HA标签的乙型脑炎病毒全长感染性克隆及其制备方法,并公开了其应用。本发明通过在NS1与NS2A之间插入HA标签,通过HA标签来指示NS2A,同时为避免影响NS2A荧光信号的观察,对NS2A第30个氨基酸进行点突变,以干扰NS2A的茎环结构,消除病毒复制过程的-1核糖体移码。本发明所述的携带HA标签的乙型脑炎病毒全长感染性克隆对于黄病毒的研究具有重要的价值。
本发明通过下述技术方案实现上述技术效果:
一种携带HA标签的乙型脑炎病毒全长感染性克隆,其制备方法具体包括下述步骤:
1)pSH7-2332-4497-HA/NS2A质粒的构建
设计两组含有HA标签的引物对并以pSH7-2332-4497为模板扩增出相互重叠度的两段位于2332-4497bp的DNA片段,得到目的片段II-1和目的片段II-2,其中引物对1的上游引物如SEQ.No.1所示,引物对1的下游引物如SEQ.No.2所示,引物对2的上游引物如SEQ.No.3所示,引物对2的下游引物如SEQ.No.4所示;将目的片段II-1和目的片段II-2利用Fusion-PCR的方法融合并与线性化的POK-12质粒连接,命名为pSH7-2332-4497-HA/NS2A。具体步骤如下:
(1)以pSH7-2332-4497为模板,扩增得到目的片段II-1和目的片段II-2,随后进行核酸电泳,胶回收,测定DNA片段浓度。其中扩增反应体系包括如下组分:Plasmid 100ng,Forward primer(10μM)1μL,Reverse primer(10μM)1μL,5×Q5 Reaction Buffer 10μL,dNTPs(10mM)1μL,Q5 DNA Polymerase 0.5μL,ddH2O补足至50μL;扩增反应条件为:98℃5min;95℃ 20s,52℃ 20s,72℃ 1min,30cycles;72℃ 5min,降温至4℃。
(2)将片段II-1与片段II-2混合,进行Fusion-PCR,其中反应体系如下:片段II-1120ng,片段II-2 120ng,5×Q5 Reaction Buffer 10μL,dNTPs(10mM)1μL,Q5 DNAPolymerase 0.5μL,ddH2O补足至50μL;扩增反应条件如下:98℃ 30s;92℃ 10s,60℃2min,12cycles;降温至4℃。
(3)向Fusion-PCR反应体系加入引物对1的上游引物(10μM)与引物对2的下游引物(10μM)各2μL,按如下反应条件进行反应:98℃ 30s;92℃ 10s,68℃ 3min,72℃ 5min,12cycles;降温至4℃,将PCR产物进行琼脂糖凝胶电泳,切胶回收,回收产物-80℃保存。
(4)将获得的PCR产物和POK-12质粒利用Not-I与Sal-I对切进行酶切处理后进行连接,转化DH5α细胞,然后涂LB平板,挑取阳性克隆测序,即可得到pSH7-2332-4497-HA/NS2A质粒。
2)pSH7-2332-4497-HA/NS2A/-ΔNS1'质粒构建
利用定点突变引物对对pSH7-2332-4497-HA/NS2A质粒上的NS2A蛋白的A30位点进行定点的突变,A30突变引物对的正向引物为SEQ.No.5所示,A30突变引物对的反向引物为SEQ.No.6所示。定点突变的方法使用现有的定点突变工艺和方法。
由于NS2A基因的起始区域存在保守的滑滑七核苷基序和RNA假结结构,通过-1核糖体移码产生NS1’(MELIAN et al.,2010),因此通过利用定点突变引物对质粒上的NS2A蛋白的A30位点进行定点的突变,消除NS1'的影响。
3)pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'的质粒构建
以pSH7-2332-4497-HA/NS2A/-ΔNS1'为模板,利用定点突变引物对质粒上的NS2A蛋白的C221位点进行定点的突变,C221位点突变引物对的正向引物为SEQ.No.7所示,C221位点突变引物对的反向引物为SEQ.No.8所示。
4)JEV全长cDNA的克隆
分别以pSH7T7-1-2361,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1',pSH7-4468-7664,pSH7-7636-10965为模板,扩增出JEV相互重叠的四个片段,再利用Fusion-PCR的方法获得病毒的基因组的全长cDNA序列,其中pSH7T7-1-2361的正向引物如SEQ.No.9所示,pSH7T7-1-2361的反向引物如SEQ.No.10所示,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'的正向引物如SEQ.No.11所示,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'的反向引物如SEQ.No.12所示,pSH7-4468-7664的正向引物如SEQ.No.13所示,pSH7-4468-7664的反向引物如SEQ.No.14所示,pSH7-7636-10965的正向引物如SEQ.No.15所示,pSH7-7636-10965的反向引物如SEQ.No.16所示。
5)体外转录
6)转染JEV RNA拯救病毒
使用DMRIE-C试剂(Invitrogen)将病毒RNA转录本转染到6孔板上的BHK细胞中。收集转染细胞及上清,在发生乙脑病毒细胞病变效应后在BHK-21细胞扩增病毒。所有从BHK-21细胞中拯救出来的重组病毒均经测序证实。含有任何不需要的突变的重组病毒的克隆体被丢弃。
本发明与现有技术相比具有如下技术优势:本发明通过在NS1与NS2A之间插入HA标签,通过HA标签来指示NS2A,同时为避免影响NS2A荧光信号的观察,对NS2A第30个氨基酸进行点突变,以干扰NS2A的茎环结构,消除病毒复制过程的-1核糖体移码。本发明所述的携带HA标签的乙型脑炎病毒全长感染性克隆对于黄病毒的研究具有重要的价值。
附图说明
图1含HA标签重组病毒结构示意图。
图2 JEV基因分段PCR扩增电泳图。
图3 JEV全长cDNA扩增电泳图;
图4拯救病毒致BHK-21细胞病变;
图5为NS2A突变和C221S突变测序鉴定结果,其中NS2A突变鉴定结果如图5-a所示,C221S突变鉴定结果如图5-b所示。
图6拯救病毒Western-Blot鉴定
图7重组病毒生长曲线比较
图8重组病毒的空斑形成试验
具体实施方式
以下通过具体实施例进一步描述本发明,但所述实施例并不以任何方式限定本发明专利保护的范围。
实施例1 JEV基因组分段扩增与克隆
通过Fusion-PCR方法在NS1-NS2A之间插入HA标签,成功构建pSH7-2332-4497-HA/NS2A质粒,在此基础上通过利用定点突变引物对质粒上的NS2A蛋白的A30位点进行定点的突变,构建pSH7-2332-4497-HA/NS2A/-ΔNS1'质粒。同样对NS2A C221进行定点突变,构建pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1',图2JEV基因分段PCR扩增电泳图。
实验例2病毒全长cDNA扩增
以pSH7T7-1-2361,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1'或pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1',pSH7-4468-7664,pSH7-7636-10965为模板扩增JEV的四个片段,经过两轮Fusion-PCR的方法获得含有T7启动子的病毒全长cDNA(图3)。
实验例3 JEV-HA/NS2A/WT-ΔNS1'和JEV-HA/NS2A/C221S-ΔNS1'的拯救与鉴定
将含有T7启动子的JEV-NS2A/C221S基因组全长利用体外转录试剂盒转录后,转染BHK-21细胞,然后每天观察细胞的病变情况,在转染后48~72小时即可观察到细胞病变,如图4所示,当细胞出现变圆、皱缩、脱落等特征性的病变时,即表示成功拯救出病毒粒子。
实验例4 NS2A突变和C221S突变的鉴定
将获得的重组病毒利用TRIzol法提取RNA,利用cDNA第一条链合成试剂盒TIANScriptIIRT Kit对病毒RNA进行反转录,经PCR扩增后进行DNA测序。结果显示,HA-tag成功插入到NS1-NS2A之间,图5为NS2A突变和C221S突变测序鉴定结果,显示NS2A突变(GCCAGA GCTAGG)成功(图5-a),C221S突变成功(图5-b)。成功获得rJEV-HA/NS2A,rJEV-HA/NS2A/WT-ΔNS1',rJEV-HA/NS2A/C221S-ΔNS1'重组病毒。
实施例5重组病毒粒子的间接免疫荧光(IFA)试验
为进一步验证拯救的重组病毒粒子,我们利用间接免疫荧光(IFA)试验对rJEV-HA/NS2A/WT-ΔNS1'和rJEV-HA/NS2A/C221S-ΔNS1'的NS2A、NS3蛋白进行检测。将拯救出感染BHK-21细胞,随后利用针对HA标签抗体和JEV NS3蛋白的特异性抗体进行验证,感染JEV-HA/NS2A/WT-ΔNS1'和JEV-HA/NS2A/C221S-ΔNS1'的BHK-21细胞可正常表达HA标签蛋白,表明NS2A蛋白可正常表达,细胞呈现特应性绿色荧光,而Mock组无红色荧光这进一步说明了拯救出的病毒粒子具有感染性。另外,NS3蛋白作为对照,在所有JEV病毒中均可正常表达,这进一步表明重组病毒拯救成功。
实施例6 HA标签和NS1'消除对病毒复制的影响
Western-Blot结果显示,用HA抗体指示NS2A时,在JEV-HA/NS2A/WT中出现两条条带,其中较大条带为NS1'。经过点突变消除NS2A二级茎环结构后,JEV-HA/NS2A/WT-ΔNS1'和JEV-HA/NS2A/C221S-ΔNS1'只有一条特异性条带,即NS2A,表明HA-NS1'在JEV-HA/NS2A/WT-ΔNS1'和JEV-HA/NS2A/C221S-ΔNS1'中均被消除(图6)。经过该策略,我们成功消除了NS1'对NS2A检测的影响。
为了分析引入HA标签和NS1'消除对病毒复制的影响,我们比较JEV-NS2A/WT和JEV-HA/NS2A/WT-ΔNS1'之间的病毒复制和蚀斑大小,结果表明HA标签的插入和消除NS1'的产生对病毒复制和蚀斑形态没有显著影响(图7,图8)。
SEQUENCE LISTING
<110> 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心)
<120> 一种携带HA标签的乙型脑炎病毒全长感染性克隆的制备方法
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Claims (4)
1.一种携带HA标签的乙型脑炎病毒全长感染性克隆,其制备方法具体包括下述步骤:
1)pSH7-2332-4497-HA/NS2A质粒的构建:设计两组含有HA标签的引物对并以pSH7-2332-4497为模板扩增出相互重叠度的两段位于2332-4497bp的DNA片段,得到目的片段II-1和目的片段II-2,其中引物对1的上游引物如SEQ.No.1所示,引物对1的下游引物如SEQ.No.2所示,引物对2的上游引物如SEQ.No.3所示,引物对2的下游引物如SEQ.No.4所示;将目的片段II-1和目的片段II-2利用Fusion-PCR的方法融合并与线性化的POK-12质粒连接,命名为pSH7-2332-4497-HA/NS2A;
2)pSH7-2332-4497-HA/NS2A/-ΔNS1'质粒构建:利用定点突变引物对对pSH7-2332-4497-HA/NS2A质粒上的NS2A蛋白的A30位点进行定点的突变,引物对的正向引物为SEQ.No.7所示,引物对的反向引物为SEQ.No.8所示。
3)pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'质粒构建:以pSH7-2332-4497-HA/NS2A/-ΔNS1'为模板,利用定点突变引物对质粒上的NS2A蛋白的C221位点进行定点的突变,引物对的正向引物为SEQ.No.9所示,引物对的反向引物为SEQ.No.10所示;
4)JEV全长cDNA的克隆:分别以pSH7T7-1-2361,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1',pSH7-4468-7664,pSH7-7636-10965为模板,扩增出JEV相互重叠的四个片段,再利用Fusion-PCR的方法获得病毒的基因组的全长cDNA序列;
5)体外转录:将回收的JEV全长cDNA进行体外转录,获得7-甲基鸟嘌呤加帽的病毒RNA;
6)转染JEV RNA拯救病毒:使用DMRIE-C试剂将病毒RNA转录本转染到BHK细胞中,收集转染细胞及上清,在发生乙脑病毒细胞病变效应后在BHK-21细胞扩增病毒,拯救出来的重组病毒均经测序证实,丢弃含有任何不需要的突变的重组病毒的克隆体即可得到携带HA标签的乙型脑炎病毒全长感染性克隆。
2.根据权利要求1所述的携带HA标签的乙型脑炎病毒全长感染性克隆,其特征在于,所述制备方法步骤1中具体包括如下步骤:
(1)以pSH7-2332-4497为模板,扩增得到目的片段II-1和目的片段II-2,随后进行核酸电泳,胶回收,测定DNA片段浓度。其中扩增反应体系包括如下组分:Plasmid 100ng,Forward primer(10μM)1μL,Reverse primer(10μM)1μL,5×Q5 Reaction Buffer 10μL,dNTPs(10mM)1μL,Q5 DNA Polymerase 0.5μL,ddH2O补足至50μL;扩增反应条件为:98℃5min;95℃ 20s,52℃ 20s,72℃ 1min,30cycles;72℃ 5min,降温至4℃;
(2)将片段II-1与片段II-2混合,进行Fusion-PCR,其中反应体系如下:片段II-1120ng,片段II-2 120ng,5×Q5 Reaction Buffer 10μL,dNTPs(10mM)1μL,Q5 DNAPolymerase 0.5μL,ddH2O补足至50μL;扩增反应条件如下:98℃ 30s;92℃ 10s,60℃2min,12cycles;降温至4℃;
(3)向Fusion-PCR反应体系加入引物对1的上游引物(10μM)与引物对2的下游引物(10μM)各2μL,按如下反应条件进行反应:98℃ 30s;92℃ 10s,68℃ 3min,72℃ 5min,12cycles;降温至4℃,将PCR产物进行琼脂糖凝胶电泳,切胶回收,回收产物-80℃保存;
(4)将获得的PCR产物和POK-12质粒利用Not-I与Sal-I对切进行酶切处理后进行连接,转化DH5α细胞,然后涂LB平板,挑取阳性克隆测序,即可得到pSH7-2332-4497-HA/NS2A质粒。
3.根据权利要求1所述的携带HA标签的乙型脑炎病毒全长感染性克隆,其特征在于,pSH7T7-1-2361的正向引物如SEQ.No.9所示,pSH7T7-1-2361的反向引物如SEQ.No.10所示,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'的正向引物如SEQ.No.11所示,pSH7-2332-4497-HA/NS2A或pSH7-2332-4497-HA/NS2A/-ΔNS1',pSH7-2332-4497-HA/NS2A/C221S/-ΔNS1'的反向引物如SEQ.No.12所示,pSH7-4468-7664的正向引物如SEQ.No.13所示,pSH7-4468-7664的反向引物如SEQ.No.14所示,pSH7-7636-10965的正向引物如SEQ.No.15所示,pSH7-7636-10965的反向引物如SEQ.No.16所示。
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