CN108721646B - 一种抑制病毒感染的方法及抗病毒药物 - Google Patents
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Abstract
本发明公开了一种抑制病毒感染的抗病毒药物，其特征在于通过靶向富含亮氨酸重复序列的LRRC25蛋白抑制病毒感染。通过对LRRC25在抗病毒方面的研究和大量实验，最后选定LRRC25作为治疗或药物的新靶向，通过药物的手段使LRRC25沉默或缺失能起到抗病毒感染的功能。
Description
技术领域
本发明涉及病毒感染治疗领域，特别涉及一种抑制病毒感染的方法及抗病毒药物。
背景技术
病毒感染是临床常见的疾病，有些病毒感染传播速度快，死亡率高，严重危及人体健康。目前，干预病毒感染主要有两种方法。一种是通过接种疫苗预防病毒感染，一种是通过抗病毒药物降低病毒的活性和复制能力。尽管已经存在众多药物和治疗方法，但是寻求更加安全有效的治疗病毒感染的靶点一直是健康研究的热点。
天然免疫是机体对抗病原微生物的第一道防线，模式识别受体在识别病原相关分子模式激活天然免疫反应，有效清除病原体过程中起着举足轻重的作用。作为抗病毒免疫反应的中坚力量，I型干扰素信号通路的调控对于抗病毒免疫尤为重要。
I型干扰素是由感染的细胞分泌的多肽，主要有三方面功能：(1)在被侵染的细胞或邻近细胞中激活细胞固有的抗病毒状态，限制感染因子，特别是病毒的传播；(2)I型干扰素能以平衡的方式，限制促炎因子信号通路和细胞因子的释放，从而调控天然免疫反应，促进抗原提呈和自然杀伤性细胞的功能；(3)I型干扰素可以激活适应性免疫系统，因此促进产生具有高抗原特异性的T，B淋巴细胞免疫反应和免疫记忆。I型干扰素对急性病毒感染具有重要保护作用。I型干扰素家族中IFN-α(具有多个部分同源基因)和IFN-β(只有一个基因)是研究比较清楚的且表达比较广泛的两个成员。
IFN-α和IFN-β结合的细胞表面受体称为IFN-α受体(IFNAR)。IFNAR是一个异源二聚体跨膜受体，由IFNAR1和IFNAR2两个亚基组成。IFNAR参与激活受体相关蛋白酪氨酸激酶Janus激酶1(JAK1)和酪氨酸激酶2(TYK2)，进而磷酸化胞内转录因子信号转导子和激活子STAT1和STAT2。酪氨酸磷酸化的STAT1和STAT2二聚化，然后入核，在细胞核内招募IRF9组成称为干扰素刺激基因因子3(ISGF3)的三分子复合物。ISGF3结合同源DNA序列干扰素刺激反应原件(ISREs)，因此激活ISGs的转录。经典I型干扰素信号诱导表达几百个ISGs，建立起抗病毒状态。ISG编码蛋白通过多种机制抑制病原体，包括抑制病毒转录，翻译和复制，促进病毒核酸降解或者改变细胞脂代谢反应。
RLR受体识别多种病毒并诱导干扰素的产生。RIG-I和MDA5通过共同的接头分子MAVS传递信号。RIG-I和MDA5与MAVS的相互作用使得RLRs被招募到MAVS结合的膜上，然后与下游信号分子结合形成MAVS信号体，诱导I型干扰素的产生。RIG-I信号通路在TRAFs水平分成两路，MAVS激活的TRAF2和TRAF6活化经典的IKKα/β激酶，诱导IκBα的磷酸化并通过蛋白酶体途径降解。活化的p65/p50 NF-κB二聚体被释放并入核，启动NF-κB依赖的基因的转录。而MAVS激活TRAF3后，TRAF3活化非经典IKK相关激酶TBK1和IKKi，进而磷酸化IRF3/IRF7的C端，诱导IRF3二聚化并入核激活IRF依赖的基因的转录。
发明内容
针对以上缺陷，本发明目的如何提供更为安全有效的抑制病毒感染的方法。
为了解决以上问题本发明提出了一种抑制病毒感染的方法，其特征在于通过靶向富含亮氨酸重复序列的LRRC25蛋白抑制病毒感染。
所述的抑制病毒感染的方法，其特征在于利用沉默LRRC25表达的siRNA或利用CRISPR/Cas9系统敲除LRRC25基因从而抑制LRRC25的作用；所述病毒为RNA病毒。
所述的抑制病毒感染的方法，其特征在于当选择利用siRNA抑制THP-1细胞表达LRRC25时，将所述siRNA转入THP-1细胞沉默LRRC25的表达，然后再加入VSV-eGFP使病毒侵染THP-1细胞，抑制LRRC25促进病毒感染的作用。
所述的抑制病毒感染的方法，其特征在于所述当选择利用CRISPR/Cas9系统敲除LRRC25基因从而抑制LRRC25的表达时，利用HEK 293T细胞制备靶向LRRC25基因的慢病毒；然后通过超速离心的方式浓缩病毒；将浓缩病毒侵染THP-1细胞；然后通过puromycin抗性筛选LRRC25单克隆THP-1细胞；然后用VSV-eGFP侵染LRRC25 KO THP-1细胞，抑制LRRC25的作用。
所述的抑制病毒感染的方法，其特征在于所述的抑制LRRC25的成分为靶向LRRC25mRNA的siRNA。
所述的抑制病毒感染的方法，其特征在于所述的抑制LRRC25的成分为靶向LRRC25的sgRNA及其制备的慢病毒。
一种抗病毒药物，其特征在于通过靶向富含亮氨酸重复序列的LRRC25蛋白抑制RNA病毒感染。
所述的抗病毒药物，其特征在于通过利用沉默LRRC25表达的siRNA或利用CRISPR/Cas9系统敲除LRRC25基因从而抑制LRRC25的作用。
所述的抗病毒药物，其特征在于当选择利用siRNA抑制THP-1细胞表达LRRC25时，将所述siRNA转入THP-1细胞沉默LRRC25的表达，然后再加入VSV-eGFP使病毒侵染THP-1细胞，抑制LRRC25促进病毒感染的作用；所述当选择利用CRISPR/Cas9系统敲除LRRC25基因从而抑制LRRC25的表达时，利用HEK 293T细胞制备靶向LRRC25基因的慢病毒；然后通过超速离心的方式浓缩病毒；将浓缩病毒侵染THP-1细胞；然后通过puromyc i n抗性筛选LRRC25单克隆THP-1细胞；然后用VSV-eGFP侵染LRRC25 KO THP-1细胞，抑制LRRC25的作用。
所述的抗病毒药物，其特征在于所述的抑制LRRC25的成分为靶向LRRC25mRNA的siRNA；所述的抑制LRRC25的成分为靶向LRRC25的sgRNA及其制备的慢病毒。
本发明通过对LRRC25在抗病毒方面的研究和大量实验，最后选定LRRC25作为治疗或药物的新靶向，通过药物的手段使LRRC25沉默或缺失,起到抗病毒感染的功能。
附图说明
图1是THP-1受VSV-eGFP感染后LRRC25的表达情况；
图2在HEK 293T细胞中过表达LRRC25，抑制I型干扰素信号通路，促进病毒感染的结果图；
图3用靶向LRRC25的siRNA沉默LRRC25的表达，增强抗病毒免疫反应的结果图；
图4利用CRISPR/Cas9系统在基因水平上敲除LRRC25后增强抗病毒免疫反应的结果图：其中，
图4A，在基因水平证明LRRC25 KO THP-1细胞系构建成功；
图4B，在蛋白水平证明LRRC25 KO THP-1细胞系构建成功；
图4C，在受到RNA病毒感染后，LRRC25缺失显著上调IFN-β的转录；
图4D，在受到RNA病毒感染后，LRRC25缺失显著上调IFN-β的释放；
图4E，在受到RNA病毒感染后，LRRC25的缺失显著上调下游ISGs的转录，例如IFIT2和IFIT1的转录；
图4F，在受到RNA病毒感染后，LRRC25缺失显著上调THP-1细胞的抗病毒能力。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
具体实施例1：THP-1受VSV-eGFP感染后LRRC25的表达情况。当THP-1细胞受到水疱性口炎病毒(VSV-eGFP)感染后，LRRC25的蛋白水平升高。
具体实验步骤为，将2ml的密度为10^6/ml的THP-1细胞铺到6孔板中，然后使用VSV-eGFP(MOI＝0.1)侵染THP-1细胞系，THP-1细胞在受到指定时间的感染后，收集细胞裂解液，通过免疫印迹实验检测细胞裂解物中LRRC25和β-actin(内参)的表达。图1是THP-1受VSV-eGFP感染后LRRC25的表达情况，可以看出当THP-1细胞受到VSV-eGFP感染后，LRRC25的蛋白水平升高。提示LRRC25参与了抗病毒反应的调控。
具体实施例2：过表达LRRC25可抑制I型干扰素信号通路，促进病毒感染。
将100ul密度为4*10^5/ml的HEK 293T细胞铺在96孔板上，于12h贴壁后转染EV(200ng)或LRRC25(200ng)表达质粒，同时共转ISRE-luc(25ng)或IFN-β-luc(25ng)。12h后用IC poly(I:C)LMW(5μg/ml))或SeV(MOI＝0.1)分别刺激24h或14h。然后通过酶标仪检测荧光报告素酶活性。荧光素酶报告基因检测结果显示，LRRC25能够抑制IC poly(I:C)LMW和SeV诱导的ISRE和IFN-β的活化。图2在HEK 293T细胞中过表达LRRC25，抑制I型干扰素信号通路，促进病毒感染的结果图，图2A部分是荧光素酶报告基因检测结果显示，LRRC25能够抑制IC poly(I:C)LMW(一种合成的RIG-I配体)和SeV诱导的ISRE和IFN-β的活化。证明LRRC25对RLR介导的I型干扰素信号通路具有负调控功能。图2B部分是在HEK 293T细胞中过表达LRRC25，并使用VSV-eGFP感染细胞，无论是在镜下观察细胞的感染情况还是流式检测细胞的感染百分比，结果都显示表达LRRC25能够显著抑制细胞的抗病毒反应。这一结果证明，LRRC25对RLR介导的I型干扰素信号通路具有负调控功能。此外，将500ul密度为4*10^5/ml的HEK 293T细胞铺在24孔板上，于12h贴壁后转染EV(200ng)或Myc-LRRC25(200ng)。24h后用VSV-eGFP(MOI＝0.001)侵染细胞到指定时间，然后在显微镜下观察细胞侵染情况，并通过流式分析感染细胞的百分比。结果显示，无论是在镜下观察细胞的感染情况还是流式检测细胞的感染百分比，过表达LRRC25能够显著抑制细胞的抗病毒反应。
具体实施例3：用靶向LRRC25的siRNA沉默LRRC25的表达，增强抗病毒免疫反应。
图3用靶向LRRC25的siRNA沉默LRRC25的表达，增强抗病毒免疫反应的结果图，将500ul的密度为10^6/ml的THP-1细胞铺到24孔板中，在THP-1细胞中转染LRRC25特异的siRNA(30nM)或不靶向任何蛋白的对照siRNA(30nM)。30h后收集细胞裂解液。经免疫印迹实验证明，与对照组相比，转染了LRRC25特异的siRNA后的THP-1细胞系中LRRC25的表达量明显减少。图3A，靶向LRRC25的siRNA沉默LRRC25的表达情况，证实了我们合成的siRNA具有良好的沉默效果。进一步，将100ul密度为4*10^5/ml的HEK 293T细胞铺在96孔板上，于12h贴壁后转染LRRC25特异序列的siRNA(30nM)或不靶向任何蛋白的对照siRNA(30nM)。12h后转染ISRE-luc(25ng)报告基因质粒。12h后转染poly(I:C)(5μg/ml)刺激细胞24h。通过酶标仪检测荧光报告素酶活性。结果显示，用靶向LRRC25的siRNA沉默LRRC25的表达后可显著促进内源性Poly I:C诱导的ISRE活化。为进一步证明LRRC25在宿主抗病毒免疫反应中的作用，将500ul的密度为10^6/ml的THP-1细胞铺到24孔板中，在THP-1细胞中转染LRRC25特异的siRNA(30nM)或不靶向任何蛋白的对照siRNA(30nM)。24h后用VSV-eGFP(MOI＝0.01)侵染细胞到指定时间。图3C部分是靶向LRRC25的siRNA沉默LRRC25的表达后，显著抑制VSV-eGFP对THP-1细胞的感染，与对照组相比，LRRC25沉默后能够大幅削弱病毒对细胞的侵染。
实施例4：利用CRISPR/Cas9系统在基因水平上敲除LRRC25后增强抗病毒免疫反应实验。
目前研究认为I型干扰素信号通路中IFN-β是宿主对抗病毒的最重要的细胞因子。一旦被上游信号诱导表达释放后，会与其受体IFNAR结合，激活STAT信号通路诱导表达更多的ISGs分子，建立起更强大的抗病毒免疫反应。为进一步证实LRRC25在抗病毒免疫反应中的功能，利用CRISPR/Cas9系统构建LRRC25 KO THP-1稳转株。提取LRRC25 KO THP-1稳转株的基因组DNA和蛋白，在基因水平测序和蛋白水平均验证所构建的细胞系为LRRC25 KOTHP-1细胞系(图4A，4B)。将2ml的密度为10^6/ml的THP-1细胞铺到6孔板中，用VSV-eGFP(MOI＝0.01)侵染对照组与LRRC25 KO THP-1细胞至指定时间。实时定量PCR实验与酶联免疫反应表明，LRRC25 KO THP-1细胞中IFN-β的转录和释放均有大幅度提升(图4C，4D)。此外，使用同样的方法证明了，在LRRC25KO THP-1细胞中IFIT2和IFIT1的转录也得到了明显的提高(图4E)。更重要的是，使用同样方法，即用VSV-eGFP(MOI＝0.01)侵染对照组与LRRC25 KO THP-1细胞至0-18h，显微镜下可观察到LRRC25缺失细胞中GFP信号(VSV-eGFP感染细胞)明显弱于对照组(图4F)。流式细胞术对感染细胞的定量分析，证实LRRC25的缺失可以使细胞的感染率从63.8％降到14.5％(图4F)。这些实验结果充分证明了靶向LRRC25，使LRRC25沉默或缺失能起到抗病毒感染的功能。
如上述一样应当注意，在说明本发明的某些特征或者方案时所使用的特殊术语不应当用于表示在这里重新定义该术语以限制与该术语相关的本发明的某些特定特点、特征或者方案。总之，不应当将在随附的权利要求书中使用的术语解释为将本发明限定在说明书中公开的特定实施例，除非上述详细说明部分明确地限定了这些术语。因此，本发明的实际范围不仅包括所公开的实施例，还包括在权利要求书之下实施或者执行本发明的所有等效方案。
Claims (2)
1.沉默富含亮氨酸重复序列的LRRC25 mRNA的siRNA或利用CRISPR/Cas9系统敲除LRRC25基因的sgRNA或由前述的sgRNA制备的慢病毒在制备用于抑制RNA病毒感染的抗病毒药物中的用途。
2.根据权利要求1所述的用途，其特征在于当选择利用siRNA抑制THP-1细胞表达LRRC25时，将所述siRNA转入THP-1细胞沉默LRRC25的表达，然后再加入VSV-eGFP使病毒侵染THP-1细胞，抑制LRRC25促进病毒感染的作用；当选择利用CRISPR/Cas9系统敲除LRRC25基因从而抑制LRRC25的表达时，利用HEK 293T细胞制备靶向LRRC25基因的慢病毒；然后通过超速离心的方式浓缩病毒；将浓缩病毒侵染THP-1细胞；然后通过puromycin抗性筛选LRRC25单克隆THP-1细胞；然后用VSV-eGFP侵染LRRC25 敲除的 THP-1细胞，抑制LRRC25的作用。
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