CN116948003A - Eif2b4敲除细胞系的构建及作为小rna病毒科病毒疫苗生产细胞系的应用 - Google Patents
Eif2b4敲除细胞系的构建及作为小rna病毒科病毒疫苗生产细胞系的应用 Download PDFInfo
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Abstract
本发明属于基因工程领域，具体涉及EIF2B4敲除细胞系的构建及作为小RNA病毒科病毒疫苗生产细胞系的应用。首先，本发明发现过表达宿主EIF2B4基因，能够抑制FMDV的复制，可作为靶点用于设计和筛选抑制FMDV复制的药物；其次，抑制或沉默宿主EIF2B4基因获得的宿主细胞能够促进FMDV病毒的复制，可作为FMDV病毒或病毒疫苗的生产细胞系；基于此，本发明设计了一种特异性靶向EIF2B4基因基因的sgRNA，结合CRISPR/Cas9技术实现了EIF2B4基因的敲除，获得的单克隆细胞系能够显著促进FMDV在细胞内的复制，为研究EIF2B4基因的分子机制提供了工具和材料，具有广阔的前景。
Description
背景技术
***是一种由***病毒引起的重要的感染偶蹄动物的疾病，属于口疮病毒属，***病毒(Foot-and-mouth Disease Virus，FMDV)是单股正链RNA病毒，属于小RNA病毒科***病毒属，是一种小型、非包膜病毒，包含一个约8500个碱基的单链正义RNA基因组，周围是一个二十面体的衣壳蛋白，四种结构蛋白VP1–4各有60个拷贝。该病毒抗原高度可变，由七个血清型组成，包括O型、A型、C型、亚洲1型、南非地区(SAT)1型、SAT2型和SAT3型，每个血清型中又有多个亚型。这七种血清型之间存在约60-70％的同源性，不同血清型之间和不同亚型之间几乎没有交叉保护，同一血清型内的不同亚型或分离株发生交叉免疫的程度均不相同，在许多国家被视为畜牧业的持续挑战。因此，该病被国际兽疫局视为最具传染性的猪病。
基因编辑技术是通过分子生物学方法对基因组中指定的DNA靶序列进行修饰，以实现DNA片段敲除、***或其他序列改变的操作方法。到目前为止，只有三种技术可用于靶向基因组编辑，即锌指核酸酶(ZFN)、转录激活物样效应核酸酶(TALEN)和簇状规则间隔短回文重复序列(CRISPR)-Cas(CRISPR相关蛋白)。
CRISPR-Cas(聚集的规则间隔短回文重复序列CRISPR相关)***是一种高效、快速、简便、廉价的细胞基因敲除技术，最初在细菌和古菌中被发现，为细菌和古菌提供了适应性免疫反应，是细菌和古菌防御入侵噬菌体和病毒的机制,更是原核细胞中重要的防御***，此***由Cas蛋白和CRISPR阵列组成。CRISPR-Cas9***将靶向基因组编辑方法更新为一种更方便、更有效的方式，从而通过在几乎任何生物体和细胞类型中陷入困境的双链断裂来促进熟练的基因组编辑。CRISPR/Cas9技术由于其设计简单、成本低、效率高、操作简单，还可以实现多个基因座的同时编辑，已成为近年来研究最多的基因编辑技术。
真核起始因子2B(eIF2B)包括五个亚基(α-ε；基因EIF2B1，2，3，4和5)，是eIF2的鸟嘌呤核苷酸交换因子(GEF)。eIF2B(真核起始因子2B)在蛋白质合成(mRNA翻译)及其调节中起关键作用。它充当GDP解离刺激蛋白(GDS)介导eIF2上的鸟嘌呤核苷酸交换，该因子(作为eIF2.GTP)将引发剂甲硫酰-tRNA带到核糖体以识别起始密码子在每一“轮”翻译起始期间。因此，eIF2B通常被称为鸟嘌呤核苷酸交换因子(GEF)。有助于在不同条件下(包括细胞应激)蛋白质合成的翻译起始和调节。EIF2B基因突变改变了细胞对应激的反应，从而导致临床症状恶化。EIF2B5突变是最常见的(60％)，其次是EIF2B2突变。
基于上述问题，首先，本发明意外发现通过过表达宿主EIF2B4基因，能够抑制FMDV的复制，可作为靶点用于设计和筛选抑制FMDV复制的药物；其次，本发明发现，抑制或沉默宿主EIF2B4基因获得的宿主细胞能够促进FMDV病毒的复制，可作为FMDV病毒或病毒疫苗的生产细胞系；基于此，为了进一步研究EIF2B4基因在细胞内调控病原微生物复制的分子机制，本发明设计了一种特异性靶向EIF2B4基因基因的sgRNA，所述的sgRNA能够特异性靶向EIF2B4基因，结合CRISPR/Cas9技术实现了EIF2B4基因的完全敲除，获得的单克隆细胞系能够显著促进FMDV在细胞内的复制，为研究EIF2B4基因在细胞内调控病原微生物复制的分子机制提供了研究工具和材料。
发明内容
针对上述技术问题，本发明首先发现通过过表达宿主EIF2B4基因，能够抑制FMDV的复制，可作为靶点用于设计和筛选抑制FMDV复制的药物；其次，本发明发现，抑制或沉默宿主EIF2B4基因获得的宿主细胞能够促进FMDV病毒的复制，可作为FMDV病毒或病毒疫苗的生产细胞系。具体包括以下内容：
第一方面，本发明提供了EIF2B4基因/蛋白作为靶点在筛选用于预防或治疗小RNA病毒科病毒感染的药物中的应用，所述药物以EIF2B4基因/蛋白为靶点，促进EIF2B4基因/蛋白的表达。
优选地，所述小RNA病毒科病毒为***病毒。
第二方面，本发明提供了EIF2B4蛋白或其表达促进剂在制备预防或治疗小RNA病毒科病毒感染的药物中的应用。
优选地，所述小RNA病毒科病毒为***病毒。
第三方面，本发明提供了EIF2B4基因敲除的宿主细胞作为小RNA病毒科病毒或病毒疫苗生产细胞系的应用。
优选地，所述小RNA病毒科病毒为***病毒。
第四方面，本发明提供了抑制或者沉默EIF2B4基因表达的试剂在制备小RNA病毒科病毒或病毒疫苗生产细胞系中的应用。
优选地，所述小RNA病毒科病毒为***病毒。
第五方面，本发明提供了一种特异性靶向EIF2B4基因的sgRNA，所述sgRNA包括EIF2B4-sgRNA1和/或EIF2B4-sgRNA2；
所述EIF2B4-sgRNA1的靶向序列为：GGGCTCGTACTCGACGCAGC；
所述EIF2B4-sgRNA2的靶向序列为：CGGAGCTTGCTTCTCGGCCA。
优选地，所述EIF2B4-sgRNA1由EIF2B4-sgRNA1-F和EIF2B4-sgRNA1-R退火形成的双链片段；所述EIF2B4-sgRNA2由EIF2B4-sgRNA2-F和EIF2B4-sgRNA2-R退火形成的双链片段；
EIF2B4-sgRNA1-F：5’-GGGCTCGTACTCGACGCAGC-3’：
EIF2B4-sgRNA1-R：5’-GCTGCGTCGAGTACGAGCCC-3’：
EIF2B4-sgRNA2-F：5’-CGGAGCTTGCTTCTCGGCCA-3’：
EIF2B4-sgRNA2-R：5’-TGGCCGAGAAGCAAGCTCCG-3’。
第六方面，本发明提供了上述第五方面所述的sgRNA在敲除宿主细胞中EIF2B4基因或在制备EIF2B4基因敲除细胞系中的应用。
第七方面，本发明提供了一种用于敲除宿主细胞中EIF2B4基因的试剂或试剂盒，所述试剂或试剂盒包括上述第五方面所述的sgRNA，或靶向敲除EIF2B4基因的打靶载体；所述靶向敲除EIF2B4基因的打靶载体包括上述第五方面所述的sgRNA和Cas9蛋白基因的编码序列。
第八方面，本发明提供了一种EIF2B4基因敲除的宿主细胞系的构建方法，其特征在于，所述方法为：利用CRISPR/Cas9***，使用上述第五方面所述的sgRNA敲除所述宿主细胞系中的EIF2B4基因。
第九方面，本发明提供了一种EIF2B4基因敲除的PK-15细胞系，所述PK-15细胞系的构建方法包括以下步骤：
(1)制备上述第五方面所述的特异性靶向EIF2B4基因的sgRNA；
(2)将步骤(1)制备的sgRNA退火并连接至PX459质粒，得到同时表达Cas9蛋白基因和打靶sgRNA序列的重组载体；
(3)将步骤(2)制备的重组载体转染PK-15细胞，用嘌呤霉素(puromycin)抗生素筛选获得EIF2B4基因敲除细胞系。
第十方面，本发明提供了上述第九方面所述的细胞系作为小RNA病毒科病毒或病毒疫苗生产细胞系的应用。
优选地，所述小RNA病毒科病毒为***病毒。
本发明的有益效果是：①本发明首先发现，过表达宿主EIF2B4蛋白，能够抑制小RNA病毒科病毒FMDV的复制，表明EIF2B4蛋白或其表达促进剂可作为抑制小RNA病毒科病毒复制的药物；②以EIF2B4蛋白靶点用于设计和筛选抑制小RNA病毒科病毒复制的药物；③抑制宿主细胞中EIF2B4基因的表达能促进小RNA病毒科病毒FMDV的复制，可用于制备小RNA病毒科病毒或病毒疫苗的生产细胞系；④本发明提供了一种靶向EIF2B4基因的sgRNA，所述sgRNA能够特异性靶向EIF2B4基因，结合CRISPR-Cas9技术可实现宿主细胞中EIF2B4基因的敲除，打靶准确、敲除效率高；⑤本发明提供了一种通过CRISPR-Cas9技术将所述sgRNA转染于宿主细胞，构建EIF2B4基因敲除细胞系的方法；⑥依据本发明所述方法获得的单克隆细胞系能够显著促进小RNA病毒科病毒FMDV的复制，提高小RNA病毒科病毒FMDV疫苗的生产量和抗原表达量，可作为小核糖核酸病毒科病毒或病毒疫苗的生产细胞系，具有广阔的应用前景。
附图说明
图1靶向EIF2B4基因组区域的sgRNA色谱示意图；
图2EIF2B4-KO-1细胞系基因缺失突变型分析图；
图3EIF2B4-KO-2细胞系基因缺失突变型分析图；
图4Western Blotting检测敲除细胞系中EIF2B4基因的蛋白水平结果；
图5EIF2B4基因功能缺失细胞系EIF2B4-KOs细胞活力检测结果；
图6相对定量检测FMDV在EIF2B4-WT和EIF2B4-KOs细胞中的复制情况及EIF2B4的敲除情况；
图7Western blot检测FMDV在EIF2B4-WT和EIF2B4-KOs细胞中的蛋白水平差异；
图8EIF2B4-KO细胞中***病毒的病毒滴度检测结果；
图9Western blot检测过表达EIF2B4抑制FMDV复制；
图10实时荧光定量检测过表达EIF2B4抑制FMDV复制。
实施例1 EIF2B4基因敲除PK-15细胞系的构建
利用NCBI数据库查询EIF2B4基因序列(SEQ ID NO.1所示，其编码的蛋白序列如SEQ ID NO.2所示)，根据CRISPR/Cas9设计原则，利用CRISPR软件在EIF2B4基因的第1个外显子区域的387位置和484位置分别设计两条sgRNA序列(结果如图1所示)：EIF2B4-sgRNA1和EIF2B4-sgRNA2：
所述EIF2B4-sgRNA1的靶向序列为：GGGCTCGTACTCGACGCAGC(SEQ ID NO.3所示)；所述EIF2B4-sgRNA1是由EIF2B4-sgRNA1-F(5’-GGGCTCGTACTCGACGCAGC-3’，SEQ ID NO.5所示)和EIF2B4-sgRNA1-R(5’-GCTGCGTCGAGTACGAGCCC-3’，SEQ ID NO.6所示)退火形成的双链片段；
所述EIF2B4-sgRNA2的靶向序列为：CGGAGCTTGCTTCTCGGCCA(SEQ ID NO.4所示)；所述EIF2B4-sgRNA2是由EIF2B4-sgRNA2-F(5’-CGGAGCTTGCTTCTCGGCCA-3’，SEQ ID NO.7所示)和EIF2B4-sgRNA2-R(5’-TGGCCGAGAAGCAAGCTCCG-3’，SEQ ID NO.8所示)退火形成的双链片段；
sgRNA的退火：将sgRNA上下游序列合成产物稀释至100μmol/L后，取上下游引物各22.5μL，并加入10×PCR buffer，共50μL体系按照99℃退火处理5min，使上下游引物形成双链；
PX459载体的酶切：利用BBSI内切酶酶切PX459载体，按照PX459载体5μL，BBSI 1μL、10×buffer 2μL、ddH2O 12μL，共20μL反应体系37℃酶切7h，胶回收酶切片段；
酶切载体与sgRNA的连接：T4连接酶0.5μL、10×T4连接酶buffer 0.5μL、PX459酶切片段1μL、退火后的sgRNA 2μL，ddH2O 1μL，共5μL体系，16℃连接2h；
PX459-sgRNA连接产物转化：将连接产物转化Trans5α感受态细胞，提取质粒，送南京擎科生物科技有限公司测序。使用BLAST软件对测序结果与目的基因序列进行对比，成功构建具有靶向EIF2B4基因不同外显子的sgRNA重组质粒，位置和序列信息如图1；重组质粒分别命名为：PX459-EIF2B4-sgRNA1、PX459-EIF2B4-sgRNA2。
细胞转染：根据Lipofectamine 2000(Invitrogen)的标准转染程序，将CRISPR重组质粒转染PK-15细胞系，转染24h后加入终浓度3μg/mL的嘌呤霉素筛选3天后，进行细胞计数，分别铺100个和300个细胞，一周后单个细胞克隆形成后用克隆环挑取单克隆(EIF2B4-KO-1(对应EIF2B4-sgRNA1)和EIF2B4-KO-2(对应EIF2B4-sgRNA2))，转移至48孔板，于37℃，5％CO2培养箱中，添加DMEM培养基中(10％胎牛血清和1％抗生素(penicilin-streptomycin))扩大培养。收集不同细胞克隆分别在DNA水平和蛋白水平进行鉴定；扩增产物进行Sanger测序，结果如图2和图3所示，EIF2B4-KO-1有19个碱基的缺失，EIF2B4-KO-2有79个碱基的缺失。
蛋白水平鉴定时用EIF2B4的抗体(Cat No.13662-1-AP)，利用Western blot检测EIF2B4的蛋白水平表达。结果如图4所示，EIF2B4-KO-1和EIF2B4-KO-2细胞系中均检测不到EIF2B4的蛋白表达。上述结果表明，EIF2B4基因敲除PK-15细胞系构建成功。
实施例2 EIF2B4基因敲除PK-15细胞系的活力检测
将WT型PK-15细胞、EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)消化，细胞消化后计数，调整细胞悬液浓度，每孔100μL接种至96孔板，置细胞培养箱正常培养一定时间；向96孔板的每个孔加入10μL CCK-8溶液，注意不要向孔中引入气泡。放细胞培养箱继续培养1-4h，用酶标仪测定每孔的OD450 nm，分析数据。结果如图5所示，EIF2B4基因敲除PK-15细胞系EIF2B4-KO-1和EIF2B4-KO-2与正常型PK-15细胞的细胞活力相同，表明EIF2B4基因的敲除并不影响宿主细胞的正常生长。
实施例3 EIF2B4-KO细胞系对FMDV复制的影响
为了研究敲除EIF2B4之后对FMDV蛋白水平的影响，分别将EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)和正常型PK-15细胞(WT)铺于6孔板中，待细胞长满后感染FMDV，于0，6，12h分别收取细胞，用Trizol裂解法提取细胞总RNA，测定浓度后，进行反转录：RNA，4μL；ddH2O，12μL；5×super MIXⅡ，4μL。反应程序：50℃，15min；85℃，5s；4℃，保存。qPCR用2×ChamQ SYBR qPCR Master Mix(Vazyme)，10μL；上下游引物各0.4μL；ddH2O，8.2μL；CDNA，1μL。GAPDH作为内参基因，对FMDV的相对表达水平及EIF2B4蛋白的表达情况进行定量。结果如图6所示，敲除EIF2B4之后在不同时间点显著促进FMDV复制。
为了研究敲除EIF2B4之后对FMDV蛋白水平的影响，分别将EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)和正常型PK-15细胞(WT)铺于6孔板中，待细胞长满后感染FMDV，于0，6，12h分别收取细胞，加1×SDS上样缓冲液混匀，煮沸15min，室温静置冷却，利用Western blot检测，上样20μL，与实例1中过程一样。结果如图7所示，在FMDV接毒后收取的样品中，EIF2B4基因敲除PK-15细胞系EIF2B4-KO中***病毒蛋白的表达高于野生型细胞，说明EIF2B4基因功能缺失单克隆细胞系能够显著促进FMDV的复制。
在EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)和WT细胞中分别比较病毒滴度的差异，将FMDV分别感染EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)和WT细胞，于24h分别收取细胞样品，反复冻融三次，用无血清的DMEM将获得细胞样品进行10-3～10-7倍梯度稀释，将BHK细胞铺96孔板，用各梯度稀释毒液分别接种96孔细胞培养板，每个孔100μL毒液，100μL细胞。置于37℃、5％ CO2恒温培养箱中培养，36h、48h、72h观察，并记录细胞病变(CPE)情况，根据Reed-Muench法计算扩增病毒的TCID50。结果如图8所示，在FMDV接毒后收取的样品中，EIF2B4基因敲除PK-15细胞系EIF2B4-KO(EIF2B4-KO-1和EIF2B4-KO-2)中***病毒的病毒滴度远高于野生型细胞，说明EIF2B4基因功能缺失单克隆细胞系能够显著促进FMDV的复制。
综上结果表明，EIF2B4基因敲除细胞系能够显著促进小核糖核酸病毒科病毒FMDV的复制，促进小核糖核酸病毒科病毒的复制，可用于小核糖核酸病毒科病毒疫苗的生产。
实施例4过表达EIF2B4抑制***病毒的复制
为了探究EIF2B4对FMDV复制的影响，首先构建EIF2B4真核表达质粒：利用EIF2B4基因上下游引物PCR扩增后，经过BamHI和NheI双酶切后，胶回收，将胶回收产物经T4 DNA连接酶与pcDNA3.1(-)已双酶切的载体连接，转化入Trans5α感受态细胞，挑取单克隆摇菌，提取质粒，将构建成功的质粒命名为EIF2B4-Myc真核表达质粒；之后在细胞培养板中接种适量的PK-15细胞悬液，放置于37℃、5％ CO2细胞培养箱中培养，待细胞融合率达到60％-70％时，依次转染EIF2B4-Myc真核表达质粒0、1、2μg和空白载体pcDNA3.1(-)质粒2、1、0μg，保证每个孔转染质粒的总量相同。转染24小时后，弃掉培养基，用PBS缓冲液清洗细胞2次，然后接种相同剂量的FMDV，感染12小时后，收取细胞样品。制备蛋白样品，进行Westernblot分析。结果如图9所示，FMDV蛋白丰度随着EIF2B4剂量依赖性升高而逐渐降低。另外，将收取的细胞样品提取总RNA后反转录，以GAPDH作为内参基因，用qPCR方法对FMDV mRNA水平进行定量。结果如图10所示，FMDV mRNA水平同样随着EIF2B4剂量依赖性升高而逐渐降低。上述结果均表明EIF2B4剂量依赖性的抑制FMDV复制。
上述结果表明，过表达宿主EIF2B4蛋白，能够抑制小RNA病毒科病毒FMDV的复制，表明EIF2B4蛋白或其表达促进剂可作为抑制小RNA病毒科病毒复制的药物；以EIF2B4蛋白靶点用于设计和筛选抑制小RNA病毒科病毒复制的药物；抑制宿主细胞中EIF2B4基因的表达能促进小RNA病毒科病毒FMDV的复制，可用于制备小RNA病毒科病毒或病毒疫苗的生产细胞系。
在本发明的基础上，通过其他技术手段使EIF2B4基因/蛋白过表达也能够促进小RNA病毒科病毒的复制；同样采用其他技术手段使任何宿主细胞中EIF2B4基因敲除，获得的EIF2B4基因敲除细胞系也能够作为小RNA病毒科病毒或病毒疫苗的生产细胞系。其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.EIF2B4基因/蛋白作为靶点在筛选用于预防或治疗小RNA病毒科病毒感染的药物中的应用，其特征在于，所述药物以EIF2B4基因/蛋白为靶点，促进EIF2B4基因/蛋白的表达。
2.EIF2B4蛋白或其表达促进剂在制备预防或治疗小RNA病毒科病毒感染的药物中的应用。
3.EIF2B4基因敲除的宿主细胞作为小RNA病毒科病毒或病毒疫苗生产细胞系的应用。
4.抑制或者沉默EIF2B4基因表达的试剂在制备小RNA病毒科病毒或病毒疫苗生产细胞系中的应用。
5.如权利要求1-4任一所述的应用，其特征在于，所述小RNA病毒科病毒为***病毒。
6.一种特异性靶向EIF2B4基因的sgRNA，其特征在于，所述sgRNA包括EIF2B4-sgRNA1和/或EIF2B4-sgRNA2；
所述EIF2B4-sgRNA1的靶向序列为：GGGCTCGTACTCGACGCAGC；
所述EIF2B4-sgRNA2的靶向序列为：CGGAGCTTGCTTCTCGGCCA。
7.如权利要求5所述的sgRNA，其特征在于，所述EIF2B4-sgRNA1由EIF2B4-sgRNA1-F和EIF2B4-sgRNA1-R退火形成的双链片段；所述EIF2B4-sgRNA2由EIF2B4-sgRNA2-F和EIF2B4-sgRNA2-R退火形成的双链片段；
EIF2B4-sgRNA1-F：5’-GGGCTCGTACTCGACGCAGC-3’：
EIF2B4-sgRNA1-R：5’-GCTGCGTCGAGTACGAGCCC-3’：
EIF2B4-sgRNA2-F：5’-CGGAGCTTGCTTCTCGGCCA-3’：
EIF2B4-sgRNA2-R：5’-TGGCCGAGAAGCAAGCTCCG-3’。
8.如权利要求6或7所述的sgRNA在敲除宿主细胞中EIF2B4基因或在制备EIF2B4基因敲除细胞系中的应用。
9.一种用于敲除宿主细胞中EIF2B4基因的试剂或试剂盒，其特征在于，所述试剂或试剂盒包括权利要求6或7所述的sgRNA，或靶向敲除EIF2B4基因的打靶载体；所述靶向敲除EIF2B4基因的打靶载体包括权利要求6或7所述的sgRNA和Cas9蛋白基因的编码序列。
10.一种EIF2B4基因敲除的PK-15细胞系，其特征在于，所述PK-15细胞系的构建方法包括以下步骤：
(1)制备权利要求6或7所述的特异性靶向EIF2B4基因的sgRNA；
(2)将步骤(1)制备的sgRNA退火并连接至PX459质粒，得到同时表达Cas9蛋白基因和打靶sgRNA序列的重组载体；
(3)将步骤(2)制备的重组载体转染PK-15细胞，用嘌呤霉素(puromycin)抗生素筛选获得EIF2B4基因敲除细胞系。
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