CN117106736A - 利用cbe构建三基因缺失prv毒株的方法和应用 - Google Patents
利用cbe构建三基因缺失prv毒株的方法和应用 Download PDFInfo
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Abstract
本发明公开了利用CBE构建三基因缺失PRV毒株的方法和应用,涉及生物技术领域,选择PRV病毒中gE、TK、gI三基因作为靶标位点,设计sgRNA序列,根据sgRNA上下游序列分别合成单链寡核苷酸,然后退火获得带粘性末端的双链DNA片段,将载体经限制性内切酶酶切后回收片段,再与带粘性末端的双链DNA片段连接,获得连接产物;将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;将质粒转染,对通过转染获得的病毒进行蚀斑纯化,每一轮均通过PCR扩增与测序的方法进行验证;纯化得到单克隆毒株;本发明方法效率高,对病毒基因的改变小,不引起病毒基因组出现大的结构变异,成本低。
Description
技术领域
本发明涉及生物技术领域,具体涉及利用CBE构建三基因缺失PRV毒株的方法和应用。
背景技术
伪狂犬病(Pseudorabies,PR)是由伪狂犬病毒(Pseudorabies virus,PRV)感染相关宿主引起的一种烈性传染性疾病。PRV无特定的宿主嗜性,可感染包括猪、牛、羊、狗在内的多种哺乳动物。猪是其自然宿主和主要传染源,PRV可引起仔猪严重的神经症状。PRV基因组全长约为143kb,GC含量高达70%,可划分为重复区域、独特长区段(Unique Long,UL)、独特短区段(Unique Short,US)。UL区包含胸苷激酶(thymidine kinase,TK)、gB(glycoprotein B)、gC、gH等基因;US区则主要有蛋白激酶(protein kinase,PK)、gD、gE、gI等基因。TK、gE和gI三个基因的缺失可降低PRV的毒性。
早期的基因编辑技术常利用DNA同源重组原理,包括ZFNs和TALENs,但因其设计复杂、敲除效率低、脱靶严重、成本高、可操作性差等缺点,发展受到了限制。CRISPR/Cas9系统自发现以来由于其高效、方便和应用范围广等特点,成为最受欢迎的基因改造工具,实现了基因的成功敲除。然而与ZFNs和TALENs等技术类似,CRISPR/Cas9实现基因编辑依赖于DNA双链断裂的产生,触发修复,造成非预期的碱基突变或脱靶切割及基因组的结构变异。为了克服这一不足,单碱基编辑技术的出现为实现精确高效的碱基转换带来了希望。
单碱基编辑技术是利用无核酸酶切割活性或切割单链产生缺口活性的改造型Cas蛋白和脱氨酶进行融合,依靠sgRNA准确地将脱氨酶锚定到靶位点,进行碱基脱氨,从而实现单碱基的精确编辑,极大提高了碱基编辑的精确度和效率。2016年,刘如谦教授团队研发出了碱基编辑器(Base Editor, BE),可直接、不可逆转地将DNA链上的碱基C替换为T(或者将G替换为A),无需切割DNA双链或提供同源DNA模板。研究人员将无切割DNA双链活性的dCas9通过linker序列与大鼠胞嘧啶核苷脱氨酶(rAPOBEC1)相融合,构建出第一代碱基编辑器BE1;后引入了尿嘧啶DNA糖基酶抑制剂(uracil DNA glycosylase inhibitor,UGI),构建出第二代碱基编辑器BE2;为利用细胞中的错配修复机制促进非编辑链以编辑链为模板进行修复,进一步增强点突变效率,研究人员恢复了dCas9的DNA单链切割活性(Cas9nD10A),构建出第三代碱基编辑器BE3。BE3是目前运用较为广泛的经典胞嘧啶碱基编辑器(cytosine base editor, CBE)。随着CBE编辑系统的不断优化和发展,研究人员又先后开发出BE4、SaBE4、BE4-Gam、SaBE4-Gam、BE4max和Anc BE4max,进一步提高了碱基编辑的效率。CBE系统凭借其高效精确的基因编辑效果,为相关疾病的基因治疗、动物疾病建模、微生物研究等提供了强有力的技术手段。目前CBE在PRV基因组改造中已经开始尝试,但只是涉及单个基因单一位点的简单突变,未进行一步法进行多个基因的多个位点同时编辑类似复杂突变,本发明通过优化CBE载体选择和sgRNA设计,一步实现了TK、gE和gI三个基因的六个位点均突变成功,蚀斑纯化后成功获得了三基因缺失的PRV-ΔTK/ΔgI/ΔgE-CBE毒株。
现有的ZFNs、TALENs耗时耗力使用昂贵,CRISPR/Cas9效率低,筛选困难,同时因为基因组的断裂造成结构变异的产生,新型的碱基编辑工具CBE通过引入终止密码子诱导基因表达的提前终止,可以实现沉默的目的,但产生的旁观者效应较多,同时受PAM限制,有些区域无法编辑。
发明内容
本发明的目的在于至少解决现有技术中存在的技术问题之一,提供利用胞嘧啶碱基编辑器快速构建gE、gI、TK基因缺失PRV毒株的方法和应用。
本发明的技术问题解决方案如下:
gE、gI、TK基因缺失的PRV毒株,于2023年6月30日保藏在中国典型培养物保藏中心(CCTCC),保藏编号CCTCC NO:V202375,保藏地址为中国湖北省武汉市武汉大学,命名为:猪伪狂犬病毒PRV-ΔTK/ΔgI/ΔgE-CBE,Porcine pseudorabies virus PRV-ΔTK/ΔgI/ΔgE-CBE;所述gE、gI、TK基因缺失的PRV毒株利用胞嘧啶碱基编辑器构建得到。
gE、gI、TK基因缺失的PRV毒株,其编码gE基因序列如SEQ ID NO.14所示,其编码gI基因序列如SEQ ID NO.15所示,其编码TK基因序列如SEQ ID NO.13所示;
利用胞嘧啶碱基编辑器快速构建gE、gI、TK基因缺失PRV毒株的方法,包括以下步骤:
S1:构建sgRNA骨架质粒;
选择PRV病毒中gE、TK、gI三基因作为靶标位点,利用胞嘧啶碱基编辑器设计sgRNA序列,根据sgRNA上下游序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将pU6-sgRNA-Puro-2A-EGFP载体经限制性内切酶酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得连接产物,即sgRNA表达载体;
所述胞嘧啶碱基编辑器为pCMV-hA3A-BE3-Y130F;
将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;
S2:质粒转染;
将质粒转染至Vero81细胞中,对通过转染获得的病毒进行蚀斑纯化,每一轮均通过PCR扩增与测序的方法进行验证;纯化得到的TK、gI、gE三基因缺失病毒。
作为本发明的优选方案,所述限制性内切酶为BbsⅠ酶。
作为本发明的优选方案,所述pCMV-hA3A-BE3-Y130F包括表达nCas9蛋白的质粒。
作为本发明的优选方案,所述单链寡核苷酸分别如SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3,SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.7,SEQ ID NO.8,SEQID NO.9,SEQ ID NO.10 ,SEQ ID NO.11,SEQ ID NO.12所示。
作为本发明的优选方案,所述转染前,将Vero81细胞在含有胎牛血清的培养液中进行培养,培养至对数生长期。
一种gE、gI、TK基因缺失的PRV毒株,采用如上任一所述的方法制得。
本发明公开了一种如上所述的gE、gI、TK基因缺失的PRV毒株在制备伪狂犬疫苗上的应用。
本发明的有益效果是:本发明的方法效率高,对病毒基因的改变小,不引起病毒基因组出现大的结构变异,成本低。同时PRV病毒GC含量较高,普通CBE编辑效果不佳,本发明利用适用于高GC背景的新型CBE进行PRV改造,提高编辑的成功率。再者,本发明提供一种新型一步制备PRV毒力基因TK,gI和gE沉默的方法,获得TK,gI和gE沉默的PRV野毒株。本发明提供的方法,对PRV基因组的TK,gI和gE基因进行编辑,每个基因提前引入两个终止密码子,成功拯救TK,gI和gE沉默毒。所述TK,gI和gE基因沉默毒与PRV野毒区别在于将终止密码子引入TK,gI和gE毒力基因的序列5’编码区域,从而达到提前终止基因正常转录表达。本发明还对高GC背景的病毒序列编辑进行改进,选用适合GC位点编辑的CBE,提高了编辑的成功率。
本发明运用该技术制备的TK,gI和gE基因缺失的PRV毒株不存在结构变异,只是引入TAG、TAA和TGA终止密码子,就能够实现TK,gI和gE表达的终止,而且该序列潜在的调控作用受到的影响最小,即本发明通过提前引入终止密码子,实现基因终止表达,无需大片段缺失,仅是改变几个碱基类型,做到对病毒原有调控序列影响最小,同时可以一次实现多个基因的同时缺失,无需分步进行,操作非常简单快捷。
本发明的gE、gI、TK基因缺失的PRV毒株,于2023年6月30日保藏在中国典型培养物保藏中心(CCTCC),保藏编号CCTCC NO:V202375,保藏地址为中国湖北省武汉市武汉大学,命名为:猪伪狂犬病毒PRV-ΔTK/ΔgI/ΔgE-CBE,Porcine pseudorabies virus PRV-ΔTK/ΔgI/ΔgE-CBE。
附图说明
图1为本发明CBE构建原理示意图;
图2为TK,gI和gE基因的PCR扩增结果图;
图3为 TK基因突变位点1的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图4为TK基因突变位点2的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图5为gI基因突变位点1的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图6为gI基因突变位点2的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图7为gE基因突变位点1的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图8为gE基因突变位点2的测序结果,方框内为PAM位点;横线处为hA3A-BE3-Y130的理论编辑窗口;三角处为点突变位点;
图9为PRV-ΔTK/ΔgI/ΔgE-CBE毒株的一步生长曲线;
图10为PRV-ΔTK/ΔgI/ΔgE-CBE毒株的噬斑形态大小;统计学分析采用双向t检验。“****”表示P值小于0.0001;
图11为WB检测gE、TK和gI基因表达情况。
图12为 hA3A-BE3-Y130F的编辑效果,横线处为BE3与hA3A-BE3-Y130F的碱基编辑窗口;三角处为突变位点。
具体实施方式
以下以具体实施例对本发明的技术方案作进一步地说明。
下述实施例中所用的试验材料,如无特殊说明,均为来自常规生化试剂。
pCMV-hA3A-BE3-Y130F载体:Addgene,货号:113428;
pCMV-BE3载体:Addgene,货号:73021;
pCMV-hA3A-BE3-Y130F载体中,由CMV启动子启动相关蛋白的表达;
单碱基编辑器pCMV-hA3A-BE3-Y130F表达用于点突变的胞嘧啶脱氨酶;
sgRNA骨架质粒包括结合靶序列的结合区和结合cas9蛋白的结合区。
一、构建sgRNA骨架质粒
本实施例选择gE/TK/gI三基因作为靶标位点, 设计sgRNA;
根据设计出的序列分别合成单链寡核苷酸。
表1 gE/TK/gI-sgRNA合成序列
将上述六对sgRNA上下游序列退火获得带粘性末端的双链DNA片段。
将pU6-sgRNA-Puro-2A-EGFP载体经BbsⅠ酶切后回收片段,再与上述带粘性末端的双链DNA片段连接,获得sgRNA表达载体。
单碱基编辑器pCMV-hA3A-BE3-Y130F包括Cas9(N),在本实施例中,具体可以为表达nCas9蛋白的质粒。
(1)构建双链sgRNA
下表为磷酸化退火的体系;
表2 磷酸化退火的体系
试剂 | 体系(μl) |
正向单链(100μM) | 1 |
反向单链(100μM) | 1 |
10×T4 DNA 连接缓冲液(NEB) | 1 |
T4 PNK(NEB) | 1 |
双蒸水 | 补充至10μl |
梯度退火程序如表3所示:
表3 梯度退火程序
温度 | 时间 | 梯度 |
37℃ | 30min | —— |
95℃ | 5min | —— |
95-85℃ | 1min | -5℃ |
85-75℃ | 1min | -5℃ |
75-65℃ | 1min | -5℃ |
65-55℃ | 1min | -5℃ |
55-45℃ | 1min | -5℃ |
45-35℃ | 1min | -5℃ |
35-25℃ | 1min | -5℃ |
4℃ | Hold | —— |
(2)构建表达线性化骨架载体质粒
下表(表4)为pU6-sgRNA-Puro-2A-EGFP载体的酶切体系。
表4 pU6-sgRNA-Puro-2A-EGFP载体的酶切体系
试剂 | 体积(μL) |
10×NEB 反应缓冲液 | 5 |
BbsⅠ限制性内切酶 | 1 |
pU6-sgRNA-Puro-2A-EGFP载体(2ug) | 3.5 |
双蒸水 | 40.5 |
总计 | 50 |
反应程序:上述酶切体系置于37℃水浴反应2h,得到酶切产物;
将上述酶切产物进行琼脂糖凝胶电泳检测,电泳结束后,通过胶回收试剂盒回收目的条带。
(3)构建表达sgRNA的骨架质粒
下表(表5)为连接反应体系
表5 连接反应体系
连接产物通过常规方法转化至Top10感受态细胞中,取适量菌液涂布于含氨苄青霉素的LB平板,待平板表面液体蒸发,倒置于37℃保温箱过夜培养16-18h,观察转化情况,挑取5个单克隆菌落,震荡培养后测序,选取测序成功的阳性克隆进一步扩大培养,抽提用于细胞转染的无内毒质粒,置于-20℃保存。
二、质粒转染
(1)转染前一天,用胰蛋白酶消化生长良好的Vero81细胞,将1×106细胞转接至六孔板,加入2mL含10%(v/v)胎牛血清的DMEM培养液(均购自Gibco),37℃、5%(v/v)CO2培养至对数生长期。
(2)参照Lipofectamine3000官方说明书进行细胞转染,CBE-sgRNA-TK3、CBE-sgRNA-TK4、CBE-sgRNA-gI1、CBE-sgRNA-gI2、CBE-sgRNA-gE1和CBE-sgRNA-gE2,同pCMV-hA3A-BE3-Y130F与PRV-JX/CH/2016基因组充分混匀后通过脂质体法共同转染至细胞中。转染总量为5 μg,其中六种sgRNA表达质粒各0.5 μg,hA3A-BE3-Y130F 1 μg,PRV-JX/CH/2016基因组1 μg。确认突变成功后,对通过转染获得的混合病毒进行蚀斑纯化,每一轮均通过PCR扩增与测序的方法进行验证。纯化得到的TK/gI/gE三基因缺失病毒,即为PRV-ΔTK/ΔgI/ΔgE-CBE,并于2023年6月30日保藏在中国典型培养物保藏中心,保藏编号CCTCC NO:V202375,命名为:猪伪狂犬病毒PRV-ΔTK/ΔgI/ΔgE-CBE,Porcine pseudorabies virusPRV-ΔTK/ΔgI/ΔgE-CBE,其编码gE基因序列如SEQ ID NO.14所示,其编码gI基因序列如SEQ ID NO.15所示,其编码TK基因序列如SEQ ID NO.13所示。
(3)转染48 h至96 h后观察病变,并收取点突变后的病毒液,将六孔板置于-80℃-常温冻融三次,收取病毒液,8000rpm离心5分钟,吸取上清液,取一部分提取基因测序。
(4)提取基因组DNA,使用引物TK-Full-F/R,gI-Full-F/R和gE-Full-F/R分别扩增TK,gI和gE基因全长序列,并对各个突变位点进行测序验证,测序成功后将测序结果与野生型PRV基因进行比对,以初步判断编辑是否有效。
扩增TK,gI和gE基因全长,见图2,针对所有编辑位点进行测序验证。测序结果如图3-图8所示,六个编辑位点中均成功引入了提前终止密码子。分别将TK蛋白第38位色氨酸密码子、234位谷氨酰胺密码子,gI蛋白第188位色氨酸密码子、347位谷氨酰胺密码子,gE蛋白第46位和110位的色氨酸密码子成功突变成终止密码子。
三、蚀斑纯化突变毒株
(1)病毒上清液按十倍比连续稀释10至106倍,将不同稀释度的病毒液接种于长满Vero81单层细胞的12 孔板中,每孔接种200μL,每半小时摇晃一次细胞板,孵育2h,吸弃病毒液,用无血清的DMEM培养基洗涤细胞表面3次备用。
(2)将高压灭菌的2%(w/v)低熔点琼脂糖微波加热溶解后,冷却至37℃左右与等体积含4%FBS的2×DMEM培养基充分混匀并迅速加入12孔板中,每孔加入1mL,室温条件下放置约5min,待培养基自然凝固后倒置放入二氧化碳细胞培养箱中进行培养。
(3)孵育48h,观察细胞病变及蚀斑形成情况,用10μL小枪头尽可能在低稀释度的孔内随机挑取多个单一蚀斑,将其分别吹入200μL的DMEM培养基,混匀后接种于24孔板内的Vero81细胞进行扩培。
(4)待细胞出现大量病变后,观察并收集病毒液、提取病毒DNA,利用引物扩增目的片段,根据相应的测序结果随后进行下一轮的纯化。
四、PRV突变毒株的一步生长曲线测定
将Vero81细胞以每孔5×105个接种于12孔细胞板中,12h后将突变病毒以1 MOI(MOI是指感染复数,感染时噬菌体与细菌的数量比值)的感染剂量接种细胞,37℃吸附2h,期间每30min中摇晃一次细胞板,之后弃去病毒液,用DMEM培养基清洗三次,每孔加入1mL含2%(v/v)FBS的细胞维持液,继续培养。在4h、8h、12h、24h、36h和48h六个时间点内分别收集病毒上清液,并将收集的病毒上清液暂时置于-80℃冰箱中保存。采用蚀斑法在Vero81细胞上对不同时间点收集的病毒液以蚀斑形成单位(PFU)的形式测定病毒滴度,绘制一步生长曲线,与亲本毒株进行比较。
生长曲线显示(图9),PRV-ΔTK/ΔgI/ΔgE-CBE毒株的生长曲线与亲本毒株PRV-JX/CH/2016相似,病毒整体上的增殖趋势无明显差异。两者在24h-48h进入生长平台期后,PRV-ΔTK/ΔgI/ΔgE-CBE病毒滴度始终低于亲本毒株PRV-JX/CH/2016。
五、突变株的蚀斑特性测定
将病毒接种于长满单层Vero81细胞的12孔细胞板中,吸附2h后,弃病毒液,并用无血清的DMEM培养基洗涤细胞表面3次,铺上含1.5wt%甲基纤维素的DMEM半固体维持培养基,置于细胞培养箱中继续培养。接毒后48h,在倒置显微镜下直接观察PRV的蚀斑形态,并分别拍摄突变毒株,PRV-JX/CH/2016毒株的蚀斑图片各25张,利用ZEEIS显微镜拍摄软件自带的直线测量工具测定并统计不同毒株的蚀斑直径大小,同时进行比较分析。
根据显微镜下的观察测量结果(图10),PRV-ΔTK/ΔgI/ΔgE-CBE毒株的噬斑形态与PRV-JX/CH/2016毒株相似,但是噬斑直径与亲本毒株相比显著减小(P<0.0001)。
六、遗传稳定性测定
将PRV-ΔTK/ΔgI/ΔgE-CBE株接种于Vero81细胞,连续增殖传代20次。分别取第0、5、10、15及20代的病毒液,冻融两次后离心取上清,使用病毒核酸提取试剂盒提取基因组DNA,随后使用引物TK-Full-F/R,gI-Full-F/R,gE-Full-F/R分别扩增TK,gI和gE基因CDS区域全长,相关扩增产物随后送至湖南擎科生物技术有限公司进行测序,测序结果经软件比对后,判断在三个病毒基因中引入提前终止密码子的6个位点是否能稳定传代。
提取传代后第0、5、10、15和20代的病毒基因组DNA后,PCR扩增并测序TK、gI和gE基因,测序结果显示第5、10、15和20代病毒与第0代病毒的TK、gI和gE基因序列均一致,TK、gI和gE基因处诱导的6个终止密码子均未见任何突变。以上结果表明PRV-ΔTK/ΔgI/ΔgE-CBE六个点突变位点均能在病毒传代过程中保持稳定。
七、蛋白表达的测定
(1)Western blot检测目的基因的表达是否沉默
取纯化后病毒接入Vero81细胞中,在接毒后24h收取病毒蛋白,进行Westernblot,检测TK,gI和gE基因的表达。
(2)实验结果见图11,结果表明,与野生型毒株相比,突变后的gE、TK和gI基因没有检测到该蛋白的表达,可见提前引入终止密码子可有效抑制gE、TK和gI蛋白的表达。
(3)上述结果表明,利用hA3A-BE3-Y130F碱基编辑器提前引入该基因的终止密码子后,病毒中TK,gI和gE的表达会受到抑制,从而达到较好的基因沉默的效果。
八、高GC背景的病毒序列编辑改进
根据PRV-JX/CH/2016毒株的gE基因序列及胞嘧啶碱基编辑器碱基编辑窗口(C4-C8),使用BE-Designer在线设计工具针对gE基因设计多条用于实现单碱基突变的sgRNA序列(表6),将含粘末端的双链gE-sgRNA3与线性化的pU6-sgRNA-Puro-2A-EGFP载体进行连接和转化。待测序成功后,提取用于胞嘧啶碱基编辑系统的gE-sgRNA表达载体的无内毒素质粒,并命名为CBE-sgRNA-gE3。
表6 gE-sgRNA合成序列
使用hA3A-BE3-Y130F和BE3分别进行编辑,比较其对GC序列背景下C的编辑效果。将1μg PRV-JX/CH/2016基因组DNA,1μg pCMV-hA3A-BE3-Y130F(或pCMV-BE3),1 μg CBE-sgRNA-gE3共转染至PK15细胞中。待病变后收取病毒液,抽提基因组DNA,PCR扩增gE基因并进行测序验证,观察理论编辑位点是否发生了有效的碱基突变。
测序结果显示(图12),位于BE3编辑窗口下GC序列背景下的C7未有明显突变;而位于hA3A-BE3-Y130F编辑窗口中C7处有少许重叠峰,有效地将gE蛋白氨基酸序列第235位谷氨酰胺密码子CAG突变为终止密码子TAG。以上结果表明针对PRV基因组中部分GC序列背景,hA3A-BE3-Y130F与BE3相比能进行更有效的编辑。
以上所述实施例仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本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,可根据以上描述的技术方案以及构思,还可以做出其他各种相应的改变以及变形,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
Claims (8)
1. gE、gI、TK基因缺失的PRV毒株,其特征在于,保藏在中国典型培养物保藏中心,保藏编号CCTCC NO:V202375,所述gE、gI、TK基因缺失的PRV毒株利用胞嘧啶碱基编辑器构建得到。
2.根据权利要求1所述的gE、gI、TK基因缺失的PRV毒株,其特征在于,其编码gE基因序列如SEQ ID NO.14所示,其编码gI基因序列如SEQ ID NO.15所示,其编码TK基因序列如SEQID NO.13所示。
3.利用CBE构建gE、gI、TK基因缺失PRV毒株的方法,其特征在于,包括以下步骤:
S1:构建sgRNA骨架质粒;
选择PRV病毒中gE、TK、gI三基因作为靶标位点,利用胞嘧啶碱基编辑器设计sgRNA序列,根据sgRNA上下游序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将pU6-sgRNA-Puro-2A-EGFP载体经限制性内切酶酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得连接产物,即sgRNA表达载体;
所述胞嘧啶碱基编辑器为pCMV-hA3A-BE3-Y130F;
将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;
S2:质粒转染;
将质粒转染至Vero81细胞中,对通过转染获得的病毒进行蚀斑纯化,每一轮均通过PCR扩增与测序的方法进行验证;纯化得到的TK、gI、gE三基因缺失病毒。
4.根据权利要求3所述的利用CBE构建gE、gI、TK基因缺失PRV毒株的方法,其特征在于,所述限制性内切酶为BbsⅠ酶。
5.根据权利要求3所述的利用CBE构建gE、gI、TK基因缺失PRV毒株的方法,其特征在于,所述pCMV-hA3A-BE3-Y130F包括表达nCas9蛋白的质粒。
6. 根据权利要求3所述的利用CBE构建gE、gI、TK基因缺失PRV毒株的方法,其特征在于,所述单链寡核苷酸分别如SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3,SEQ ID NO.4,SEQID NO.5,SEQ ID NO.6,SEQ ID NO.7,SEQ ID NO.8,SEQ ID NO.9,SEQ ID NO.10,SEQ IDNO.11,SEQ ID NO.12所示。
7.根据权利要求3所述的利用CBE构建gE、gI、TK基因缺失PRV毒株的方法,其特征在于,所述转染前,将Vero81细胞在含有胎牛血清的培养液中进行培养,培养至对数生长期。
8.一种如权利要求1或2所述的gE、gI、TK基因缺失的PRV毒株在制备伪狂犬疫苗上的应用。
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