CN116064531B - 一种抑制cvb5病毒复制的长链非编码rna及应用 - Google Patents
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Abstract
本发明公开一种抑制CVB5病毒复制的长链非编码RNA及应用，涉及生物医药技术领域。所述RNA为基因间长链非编码RNA LINC1392；所述LINC1392的核酸序列如SEQ ID NO.1所述。本发明所述的LINC1392定位于细胞质，具有依赖于CVB5病毒感染剂量的基本表达特征。实验表明，当敲低本发明所述的LINC1392后，CVB5病毒的复制水平较对照组有显著的升高，即本发明中所提供的LINC1392具有抑制CVB5病毒复制的作用。本发明中所提供的LINC1392序列合成后，可用于进一步制备具有预防CVB5病毒感染的药物，用于手足口病的预防和治疗。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种抑制CVB5病毒复制的长链非编码RNA及应用。
背景技术
柯萨奇病毒B组5型（Coxsackie virus B5, CVB5）是导致手足口病（Hand-foot-and-mouth disease, HFMD）的重要病原体之一，主要感染5岁以下婴幼儿。CVB5病毒的主要传播途径是粪-口途径，在肠道中增殖，随后通过血液循环到达到其他组织器官，进而引发疾病。临床症状通常表现为发热、黏膜皮疹和疱疹等，病情严重者会导致神经系统并发症，甚至死亡。然而，目前尚无针对CVB5感染引起HFMD和神经系统疾病的治疗手段和有效疫苗。
长链非编码RNA（long non-coding RNA, lncRNA）是转录本长度大于200个核苷酸，不具有蛋白编码功能的RNA分子，在众多生命活动中发挥着重要作用。随着高通量测序技术的发展，lncRNA被证明能够通过多种调控机制参与病毒感染，在宿主抗病毒免疫反应中发挥至关重要的作用。研究报道，lncRNA作为新型调控因子，不仅可以通过影响细胞因子和趋化因子的表达、表观遗传修饰作用调控病毒复制，还可以调控干扰素信号通路、NF-κB信号通路和Wnt信号通路等免疫相关通路发挥作用，进而调控病毒复制。
目前，lncRNA在CVB5病毒复制中的调节机制仍未有研究报道，因此研究一种具有抑制CVB5病毒复制功能的宿主lncRNA，对于CVB5病毒致病机制的理解和抗CVB5病毒感染药物的研发具有重要的意义。
发明内容
本发明的目的在于，提供一种抑制CVB5病毒复制的长链非编码RNA LINC1392，具有抑制CVB5病毒复制功能，用于抗CVB5病毒药物的研发。
为实现上述技术目的，达到上述技术效果，本发明是通过以下技术方案实现：
一种抑制CVB5病毒复制的长链非编码RNA，所述RNA为基因间长链非编码RNALINC1392，所述LINC1392的核苷酸序列如SEQ ID NO.1所述。
本发明的另一目的在于，提供一种检测LINC1392表达的方法，包括以下步骤：
S1：样品及对照样品利用Trizol法提取总RNA并进行定量；
S2：样品选取0.1μg总RNA进行实时荧光定量PCR，将RNA作为荧光定量PCR模板，荧光定量PCR的上游引物如SEQ ID NO.2所示；荧光定量PCR的下游引物如SEQ ID NO.3所示，采用SYBR-Green荧光定量试剂盒进行荧光定量检测。
本发明的另一目的在于，提供一种LINC1392在制备抗CVB5病毒复制药物中的应用。
本发明的另一目的在于，提供一种LINC1392在制备预防或治疗手足口病的药物中的应用。
本发明的有益效果：
本发明抑制CVB5病毒复制的长链非编码RNA LINC1392，当敲低本发明中LINC1392后，CVB5病毒复制水平较对照组有显著升高，即本发明中所提供的LINC1392具有抑制CVB5病毒复制的作用。
本发明中所提供的LINC1392序列，合成后可用于进一步制备出具有抑制CVB5病毒复制的药物，用于手足口病的预防和治疗。
当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有优点。
附图说明
图1为本发明实施例所述CVB5病毒感染组和空白对照组中LINC1392表达量的高通量测序结果；
图2为本发明实施例所述荧光定量PCR检测LINC1392的表达差异；
图3为本发明实施例所述荧光定量RCR检测LINC1392的细胞定位；
图4为本发明实施例所述荧光定量PCR检测不同剂量CVB5病毒感染时LINC1392的表达；
图5为本发明实施例所述荧光定量PCR检测CVB5病毒感染几种不同细胞时LINC1392的表达；
图6为本发明实施例所述Western Blot检测LINC1392敲低时与对照组相比CVB5病毒的复制水平；
图7为本发明实施例所述病毒TCID50测定验证LINC1392敲低时与对照组相比CVB5病毒的复制水平。
具体实施方式
为了更清楚地说明本发明实施例的技术方案，下面将结合附图对实施例对本发明进行详细说明。
实施例1
本发明采用高通量测序方法对CVB5病毒感染组与空白对照组中LINC1392表达量进行检测，具体包括以下步骤：
将1MOI的CVB5病毒接种于提前铺好的6孔板SHSY5Y细胞中，空白对照组加入相同量的无菌PBS，37℃、CO2培养箱中继续培养24小时后，采用Trizol法（Trizol提取试剂购自Taraka生物公司）提取样品的总RNA。随后通过RNA-seq技术筛选出CVB5病毒感染组和对照组中具有显著差异的长链非编码RNA LINC1392。
本发明中，LINC1392差异倍数log2 Fold Change为10.37，p<0.01（如图1所示）。
实施例2
如图2-5所示
所述LINC1392的核苷酸序列如SEQ ID NO.1所述，LINC1392全序列由北京由北京诺禾致源科技股份有限公司提供。
本发明采用荧光定量PCR（RT-PCR）对LINC1392在CVB5感染后的表达水平进行检测。具体步骤如下：
每样品选取0.1μg总RNA采用一步法反转录试剂盒One Step TB Green®PrimeScript™ RT-PCR Kit (Takara生物公司) 对各组的总RNA进行定量，反应体系和反应程序按照说明书进行，以GAPDH为内参计算LINC1392相对表达量。
将RNA作为荧光定量PCR模板，荧光定量PCR的上游引物如SEQ ID NO.2所示；荧光定量PCR的下游引物如SEQ ID NO.3所示，采用SYBR-Green荧光定量试剂盒进行荧光定量检测。
同时，采用荧光定量PCR技术检测了LINC1392的细胞定位以及表达依赖于不同剂量CVB5病毒感染，且在不同细胞中的表达特性。
实施例3
如图6-7所示
在本发明中，LINC1392能够抑制CVB5病毒的复制。
本发明采用siRNA技术敲低LINC1392的表达，用于敲低LINC1392的siRNA的核苷酸序列如SEQ ID NO.4所示。
具体的，取100μL的无血清培养基和5μL的siRNA溶液混合，得到混合液A；取100μL的无血清培养基和10μL的Lipofectamine-2000 Reagent混合，室温静置5min，得到混合液B。将AB液混合，室温静置孵育20min后，加入提前铺好的6孔板细胞中，同时设置对照组，于37℃、5%CO2培养箱中继续培养24h。随后提取各孔中收集的细胞总RNA，实时荧光定量PCR检测细胞中LINC1392的表达水平，表达差异倍数为3。
另外，本发明采用TCID50滴度测定实验检测了LINC1392敲低时CVB5病毒滴度，与对照组相比，实验组病毒滴度上升了1.5倍lgTCID50。同时，Western Blot实验检测了LINC1392敲低时，CVB5结构蛋白VP1的表达量显著提高。
需要说明的是：本发明实例中CVB5病毒，SHSY5Y细胞为自行保存，无血清培养基购自Hyclone公司，胎牛血为AusGeneX澳洲胎牛血清。
在本说明书的描述中，参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。
Claims (3)
1.一种抑制CVB5病毒复制的长链非编码RNA，其特征在于：所述的长链非编码RNA为LINC1392，所述LINC1392的核苷酸序列如SEQ ID NO.1所述。
2.如权利要求1所述抑制CVB5病毒复制的长链非编码RNA在制备抗CVB5病毒复制药物中的应用。
3.如权利要求1所述抑制CVB5病毒复制的长链非编码RNA在制备治疗手足口病的药物中的应用。
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