CN110117577A - 低毒单纯疱疹病毒系统及其构建方法和应用 - Google Patents
低毒单纯疱疹病毒系统及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了单纯疱疹病毒1型H129临床毒株衍生的重组低毒单纯疱疹病毒系统及其构建方法和应用。利用本发明的靶向载体构建的重组病毒,是显著减毒的H129重组病毒,外源基因表达丰度非常高,不论是在体外或是动物活体测试中都展现出低毒性、长时程高表达的特性,中枢感染动物不会致病,很多脑区神经元被高亮标记,神经元胞体、轴/树突纤维和树突棘等精细结构标记清晰。低毒单纯疱疹病毒适于做目的基因长效高表达的基因转导载体,神经环路长时程结构示踪,因其低毒性也适用于功能神经环路解析;另外低毒HSV在神经系统靶向基因治疗、病毒复制与致病机理分析、动物感染模型建立、抗病毒药物筛选、溶瘤治疗等方面具有广泛的应用价值。
Description
技术领域
本发明属于生物技术领域,涉及神经生物学及病毒学,具体涉及低毒单纯疱疹病毒示踪系统及构建方法和应用,利用本发明提供的靶向载体构建的重组单纯疱疹病毒可用于神经环路标记、基因转导、溶瘤治疗、动物感染模型建立、病毒复制与致病机理的分析、抗病毒药物筛选等方面。
背景技术
病毒跨突触示踪技术在近年来神经回路解析中得到了越来越广泛的应用。传统的神经网络示踪方法,如染料、化合物示踪剂、蛋白肽类等,能沿轴突转运但因不能跨突触,只能标记局部神经元形态。相对嗜神经病毒作为示踪工具有明显优势:1)高效感染神经细胞;2)能跨突触传播;3)跨突触方向可控,可特异逆向或顺向传播;4)病毒跨突触后能自我复制,信号不衰减;5)可携带复杂调控元件和多样标示物等。
目前常用的嗜神经病毒主要有来源于α疱疹病毒科的伪狂犬病毒(Pseudorabiesvirus,PRV)和单纯疱疹病毒(Herpes simplex virus,HSV)以及棒状病毒科的狂犬病毒(Rabies virus,RV),其它还有水泡性口炎病毒(vesicular stomatitis virus,VSV)等,其中PRV Ba rtha株和RV是逆向跨突触感染,VSV两个方向都可以传播,相对能特异顺行跨突触传播的是HSV-1H129株,和神经冲动传递的方向一致,非常适合于标记输出神经网络。不过目前HSV工具病毒普遍存在毒性大、灵敏度低的缺陷,H129是从一个脑炎患者脑部分离的毒株,野生株具有较大的毒性,感染的细胞一般两天内很快凋亡;小鼠经脑感染H129后一般只能存活3-5天,无法开展长时程神经环路结构示踪,更谈不上用于功能环路解析,因此开展对HSV H129的减毒、增敏研究很有必要。
单纯疱疹病毒1型(HSV 1)是一种普遍分布、条件致病的病原体,是大的具囊膜病毒,直径约200nm,核心为双链DNA基因组,约153kb,由共价连接的长片段(UL)和短片段(US)组成,每一片段均含单一序列和反转重复序列,因此可以形成4种同分异构体(见图1A)。本发明为实现对H129显著减毒,选择敲除病毒的神经毒力因子基因γ34.5。γ34.5基因在HSV基因组中有两个拷贝,分别位于UL长片段的末端重复序列TRL和IRL中,基因全长1007bp(序列为SEQ ID NO.2所示),编码具有多功能的ICP34.5蛋白:机体细胞被病原体或病毒感染后很快会激活固有免疫应答,包括诱导干扰素及其它重要抗病毒蛋白的产生。病毒感染使真核细胞翻译起始因子eIF2α磷酸化,会导致细胞蛋白合成终止,从而起到阻抑病毒复制的效果。而HSV为复制自身进化出多样策略逃避宿主防御机制,ICP34.5通过与宿主蛋白磷酸酶1(PP1)结合形成复合物,调控介导eIF2α的去磷酸化,从而保持宿主细胞蛋白的合成代谢,服务于病毒复制过程。ICP34.5另外还具有拮抗干扰素诱导产生抗病毒蛋白、拮抗宿主细胞自噬清除病毒等功能。
发明内容
本发明的目的在于提供了一种低毒单纯疱疹病毒示踪系统,所述的低毒HSV通过将H SV-1H129基因组中两个拷贝的神经毒力因子γ34.5基因完全敲除,将完整的荧光基因表达盒插入敲除的γ34.5基因座位中得到。
本发明的另一个目的在于提供了一种制备携带红色荧光基因的低毒单纯疱疹病毒所用的靶向载体,序列为SEQ ID NO.1所示。
本发明还有一个目的在于提供了低毒单纯疱疹病毒示踪系统的应用,所述的应用包括:神经环路标记示踪、大容量基因转导载体、神经系统靶向基因治疗、病毒复制与致病机理分析、动物感染模型建立、抗病毒药物筛选、溶瘤治疗等。
为了实现以上目的,本发明采用如下技术方案:
低毒单纯疱疹病毒示踪系统,通过下述方法得到:将HSV-1H129基因组中神经毒力因子γ34.5两个拷贝基因完全敲除,将完整的荧光基因表达盒插入敲除的γ34.5基因座位中得到,所述的荧光基因表达盒包括hUbC启动子、荧光基因和WPRE片段。
得到的低毒单纯疱疹病毒示踪系统包括以下特征:
1)H129基因组中神经毒力因子γ34.5两个拷贝基因被完全敲除;
2)完整的外源基因表达盒插入敲除的γ34.5基因座位,并且转录方向保持一致;
3)外源基因表达盒:本发明所用荧光蛋白表达盒包含高效启动转录的启动子(泛素启动子hUbC)、荧光蛋白基因、基因下游引入土拨鼠肝炎病毒转录后调控元件(WPRE)增强外源基因表达;
4)γ34.5基因的启动子位于其上游末端重复序列中,我们构建了绿色荧光基因前不加外源启动子的低毒HSV,绿色荧光仍能有效表达,说明γ34.5基因上游的启动子在低毒单纯疱疹病毒高灵敏表达中发挥促进作用。
以上所述的靶向载体,具体是通过下述方法得到:
(1)同源臂克隆构建
敲除双拷贝γ34.5基因,以HSV-1H129基因组序列为模板,设计引物克隆γ34.5基因上游同源臂(UHA,长527bp,GC含量80%),下游同源臂(DHA,长547bp,GC含量66%);克隆的上下游同源臂片段分别通过Hind III及BamH I、BamH I和XbaI酶切后连入pcDNA3.1+载体,命名为pH129Δγ34.5,该载体在克隆的上下游同源臂中间引入一个多克隆位点接头,引入的6种内切酶分别是-AgeI-ClaI-HpaI-BamHI-EcoRI-SwaI-PacI-SbfI-,便于后续克隆外源基因或表达控制元件插入靶向载体。
(2)外源基因表达盒质粒构建
以FUGW质粒为模板,分别克隆hUbC启动子、WPRE片段,经酶切连入pcDNA3.1(+)载体获得pcDNA3.1-hUbC-WPRE载体;克隆红色荧光基因(tdTomato)或绿色荧光基因(E GFP)片段,通过KpnI、XbaI酶切位点连到pcDNA3.1-hUbC-WPRE载体的hUbC和WPR E中间,获得外源基因表达盒质粒。
(3)携载荧光基因表达盒的重组靶向载体构建
设计引物从荧光基因表达盒质粒上PCR克隆获得hUbC-tdTomato-WPRE-PA或hUbC-E GFP-WPRE-PA整个表达盒DNA,经EcoR I和Sbf I双酶切后连入pH129Δγ34.5,构建得到重组靶向载体pH129Δγ34.5-hUbC-tdTomato-WPRE-PA或靶向载体pH129Δγ34.5-hUbC-EG FP-WPRE-PA。
利用上述方法,得到制备表达tdTomato红色荧光(SEQ ID NO.3)的低毒单纯疱疹病毒的靶向载体,序列为SEQ ID NO.1所示。
利用上述方法,得到制备表达EGFP绿色荧光(SEQ ID NO.4)的低毒单纯疱疹病毒的靶向载体,序列为SEQ ID NO.5所示。
低毒单纯疱疹病毒示踪系统的应用,所述的应用包括:神经环路标记示踪、大容量基因转导载体、神经系统靶向基因治疗、病毒复制与致病机理分析、动物感染模型建立、抗病毒药物筛选、溶瘤治疗等。
本发明与现有技术相比,具有以下优点和效果:
(1)为实现HSV H129显著减毒,选择克隆基因组TRL和IRL重复序列中完全相同的γ34.5上下游同源臂构建靶向载体,保证将H129基因组中两个拷贝的神经毒力基因γ34.5都完全敲除;
(2)外源基因表达盒插入敲除的γ34.5基因座位,并且转录方向和原先γ34.5基因保持一致,这样设计是因为γ34.5上游同源臂含有启动子序列,和重组引入的同向的外源启动子一起将显著增强外源基因的表达强度和效力;
(3)建立了灵活、高效的HSV重组靶向载体构建系统,通过克隆同源臂并引入多克隆位点接头,便于后续分子遗传操作插入外源基因或多个转基因;
(4)本发明提供的低毒单纯疱疹病毒毒性很小,中枢感染动物不致病,标记神经元形态良好,而且荧光标记强度高,神经元胞体、轴树突纤维和树突棘等精细结构可视化标记清晰,可进行长时程神经环路结构示踪,也真正适用于功能神经环路解析;
(5)本发明获得的低毒单纯疱疹病毒外源基因表达盒选择荧光蛋白基因只是作为一个范式,因此还可以用其它任何目的外源基因替代本发明用的荧光蛋白基因构建定制表达目的基因的低毒单纯疱疹病毒。
(6)本发明获得的低毒单纯疱疹病毒系统感染宿主范围广,所述的应用对象不仅限于啮齿类动物大、小鼠,还适用于斑马鱼、雪貂、树鼩、非人灵长类猴等动物的脑科学研究、基因转导、溶瘤等领域。
附图说明
图1为HSV基因组结构示意图;
图1中A:HSV H129野生型毒株基因组的结构示意图。两个γ34.5基因分别位于UL末端重复序列TRL和IRL的“a”序列中;
图1中B:γ34.5基因双敲除、hUbC启动子驱动红色荧光蛋白表达的低毒HSV(H129Δγ34.5-hUbC-tdTomato-WPRE)的基因组结构示意图。双拷贝γ34.5基因转录方向相反,见图中箭头标示;
图1中C:γ34.5基因双敲除、未加外源启动子表达绿色荧光蛋白的低毒HSV(H129Δγ34.5-EGFP-WPRE)的基因组结构示意图。
图2为低毒单纯疱疹病毒重组、挑斑纯化及感染细胞荧光表达图;
图2中A:表达红色荧光的低毒H129Δγ34.5-hUbC-tdTomato-WPRE的重组及纯化;
图2中B:表达绿色荧光的低毒H129Δγ34.5-hUbC-EGFP-WPRE的重组及纯化。
图3为低毒单纯疱疹病毒基因组分子鉴定图。
图4为低毒单纯疱疹病毒体外感染原代培养神经元;
A,B:神经元微流控三腔培养体系示意图;
C:连续观察低毒单纯疱疹病毒感染的神经元状态良好、能存活至少2周。
图5为低毒单纯疱疹病毒高效示踪VTA神经网络。
图6为低毒单纯疱疹病毒高效示踪前嗅核神经网络。
图7为低毒单纯疱疹病毒长时程(1月)、高效示踪VTA神经网络。
图8为低毒单纯疱疹病毒长时程(2月)、高效示踪初级运动皮层M1神经网络。
图9为低毒单纯疱疹病毒活体标记毒性测试。
图10为无外源启动子的重组低毒单纯疱疹病毒的构建及制备。
具体实施方式
本发明所述技术方案,如未特别说明,均为本领域的常规技术。所述试剂或材料,如未特别说明,均来源于商业渠道。
可以通过引用本发明以下的某些具体实施例的详细描述而更容易的理解本发明。
提供如下实施例的目的是说明本发明的原理,它们决不旨在限制或缩小本发明的范围。
实施例1:
低毒单纯疱疹病毒重组靶向载体构建
(1)同源臂克隆
在NCBI GenBank数据库中查询HSV-1H129全基因组序列(GenBank:GU734772.1),分析神经毒力基因γ34.5及其侧翼DNA序列。γ34.5基因在HSV基因组中有两个拷贝,分别位于UL长片段的末端重复序列TRL和IRL中,基因全长1007bp,序列为SEQ ID NO.2所示,GC含量高达80%;双拷贝γ34.5基因转录方向相反,见图1B中箭头标示;本发明设计敲除γ34.5全长基因(1007bp),抽提纯化HSV-1H129病毒基因组DNA,以其为模板,设计引物克隆γ34.5基因上游同源臂(UHA,长527bp,GC含量80%),所用引物序列为UHA-F:5'CCCAAGCTTAGCCCGGGCCCCCCGCGGGC 3'(SEQ ID NO.6)';UH A-R:5'CGGGATCCGTTAACCCATCGATGGACCGGTGGAGACAGAGAGCGTGCCGG 3'(SEQ ID NO.7);下游同源臂(DHA,长547bp,GC含量66%),PCR所用引物序列为D HA-F:5'CCGGAATTCATTTAAATCCTTAATTAAGGCCTGCAGGAACTTGCAAGAGGCCT TGTTC 3'(SEQ ID NO.8)';DHA-R:5'GCTCTAGAACCCCACGCCTTTCCCCTCC 3'(SEQ ID NO.9)。
PCR反应体系为50ul,其中模板DNA一般用50-100ng,5×PrimeStar HS buffer 10μl,20μM/μl引物1、引物2各0.7μl,dNTPs 5μl,Prime star HS高保真酶0.6μl,补灭菌水到50μl。PCR扩增条件为:98℃5min,(98℃30s,60℃30s,72℃1min/kb)循环32次,72℃延伸10min,16℃30min。克隆的上下游同源臂片段分别通过Hind III及Bam H I、BamH I和XbaI酶切后连入pcDNA3.1+载体,命名为pH129Δγ34.5。
(2)外源基因表达盒质粒构建
以FUGW质粒为模板,分别克隆hUbC启动子、WPRE片段;将克隆带有NheI、KpnI酶切位点的泛素启动子片段和带有XbaI、ApaI酶切位点的转录增强元件WPRE,经酶切连入pcDNA3.1(+)载体获得pcDNA3.1-hUbC-WPRE载体;克隆红色荧光基因(tdTomato)或绿色荧光基因(EGFP)片段,通过KpnI、XbaI酶切位点连到pcDNA3.1-hUbC-WPRE载体的hUbC和WPRE中间,获得外源基因表达盒质粒,经酶切、测序证明构建正确。该构建中设计使用的引物及序列见下表。
(3)携载荧光基因表达盒重组靶向载体构建
设计引物从荧光基因表达盒质粒上PCR克隆获得hUbC-tdTomato-WPRE-PA或hUbC-E GFP-WPRE-PA整个表达框,所用引物为UT/GWPA-F:5'AGTCCAGTGTGGTGGAATTCGCGCCGGGTTTTGGCGCCTC 3'(SEQ ID NO.16)和UT/GWPA-R:5'CTCTTGCAAGTTCCTGCAGGCCATAGAGCCCACCGCATCC 3'(SEQ ID NO.17)。切胶回收纯化荧光基因表达框大片段经EcoR I和Sbf I双酶切后连入前期构建好的敲除γ34.5全长基因的靶向载体p H129Δγ34.5,经酶切、测序证明荷载荧光基因的靶向载体构建成功,得到重组靶向载体pH129Δγ34.5-hUbC-EGFP-WPRE-PA和靶向载体pH129Δγ34.5-hUbC-tdTomato-WPRE-PA,pH129Δγ34.5-hUbC-tdTomato-WPRE-PA靶向载体分子构建示意图见图1中B,其全序列见SEQ IDNO.1。携带绿色荧光基因前无外源启动子的低毒单纯疱疹病毒重组用靶向载体pH129Δγ34.5-EGFP-WPRE-PA构建示意图见图1中C所示,序列为SEQ ID NO.5所示。
实施例2:
低毒单纯疱疹病毒重组、挑斑纯化及扩增制备
①病毒的重组:抽提靶向载体pH129Δγ34.5-hUbC-tdTomato-WPRE和pH129Δγ34.5-hUb C-EGFP-WPRE-PA,采用脂质体转染的方法转染293T细胞,6小时后更换含2%FBS的维持培养基并加入单纯疱疹病毒H129株进行感染;在不同时间观察荧光的表达和细胞病变情况,待细胞全部病变后收集细胞培养基上清置于-80℃冰箱。
②病毒的纯化:将收集的病毒上清经三次反复冻融、6500g离心10分钟去净细胞碎片,吸取10μl病毒上清感染Vero细胞,1天后观察感染细胞有无荧光表达来确定新型病毒是否重组成功;后期取重组成功的病毒上清连续10倍梯度稀释后感染Vero细胞,吸附1小时后铺琼脂(含5%胎牛血清的DMEM培养基和2%琼脂1:1混合)。48-72hr后待病毒斑形成后,在倒置荧光显微镜下进行挑斑;新型重组病毒经过6轮左右的挑斑纯化将野生型病毒去除,获得纯化的新型重组病毒H129Δγ34.5-hUbC-tdTomato-WPRE(缩写为H129LT-tdT)和H129Δγ34.5-hUbC-EGFP-WPRE-PA(缩写为H129LT-EGFP)。低毒单纯疱疹病毒重组、挑斑纯化及感染细胞荧光表达情况见图2,可见两种低毒HSVLT的荧光表达很强、很亮。
实施例3:
低毒单纯疱疹病毒基因组分子鉴定
取浓缩纯化的野生型H129以及低毒HSVLT(H129Δγ34.5-hUbC-tdTomato-WPRE)病毒在100℃灭活10分钟,后续用于γ34.5基因的分子鉴定。选择γ34.5 726bp ORF片段设计引物,鉴定所用引物及序列为γ34.5-F:5'ATGGCCCGCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGG 3'(SEQ ID NO.18);γ34.5-R:5'TTAGACCGAGTTCGCCGGGCCGGCTCCGCGGGCCAGGGCCCGGGC 3'(SEQ ID NO.19)。由于γ34.5基因GC含量高达82%,选用高GC buffer体系扩增γ34.5ORF:PCR反应体系为50ul,其中灭活HSV病毒样用量为1-5μl,2×PrimeStar high-GC buffer 25μl,20μM/μl引物1、引物2各0.7μl,dNTPs 5μl,Primestar HS高保真酶0.6μl,补灭菌水到50μl。PCR扩增条件为:98℃5min,(98℃30s,60℃30s,72℃1min)循环32次,72℃延伸10min,16℃30min。分子鉴定结果如图3所示,阴性对照无条带,阳性对照为构建的表达γ34.5的质粒(pcDNA3.1-hUbC-γ34.5)经PCR扩增出一条长700bp左右的目的条带;浓缩的野生型H129病毒也扩增出同样大小的目的条带,只是条带稍淡;而低毒HSVLT(H129Δγ34.5-hUbC-tdTomato-WPRE)多次重复实验无论选用1μl、3μl、5μl浓缩病毒进行PCR均未扩增出700bp左右的目的条带,结果说明低毒HSVLT基因组中双拷贝γ34.5基因已被完全敲除。
实施例4:
低毒单纯疱疹病毒体外感染培养神经元
利用微流控神经元培养芯片测试低毒单纯疱疹病毒的感染特性及毒性,微流控芯片装置分为三腔:胞体侧(soma)、微通道和轴突侧(axon)(如图4中A所示),为了控制轴突在微通道中定向生长,胞体侧液面始终高于轴突侧,保证胞体侧维持较高的压力差。将制备的表达红色荧光的低毒HSV(H129Δγ34.5-hUbC-tdTomato-WPRE)按MOI=1加在轴突端感染三腔培养的小鼠原代神经元,连续观察感染后神经元能够存活两周以上时间,而且神经元状态良好,荧光表达标记清晰、能观察到持续生长的树突纤维(见图4中C),而野生型HSV感染离体培养的神经元第2天就能观察到树突和轴突纤维的片段化损伤、逐渐萎缩消除,说明低毒H129LT毒性较野生型H129显著降低。
实施例5:
低毒单纯疱疹病毒高效示踪VTA神经网络
将制备的表达红色荧光的低毒HSV进行动物活体测试。精确量取200nl实施例2制备的H129Δγ34.5-hUbC-tdTomato-WPRE(病毒滴度是2×109PFU/ml),立体定位注射C57BL/6小鼠大脑腹侧被盖区(VTA)脑区,小鼠未表现感染症状,待感染14天后麻醉动物,分别用0.9%(V/V)生理盐水灌流,然后用4%(V/V)多聚甲醛灌流;取出脑组织浸泡于4%(V/V)多聚甲醛液中,然后将脑组织先置于20%(V/V)蔗糖溶液中1天,接着置于30%(V/V)蔗糖溶液中2天;切片前将脑组织底部切平,置于底座上包埋冰冻1小时后切片;挑取脑片使用荧光显微镜观察。
野生型H129感染小鼠第二天就出现感染症状,一般5天内死亡;而低毒HSVLT感染小鼠无明显感染症状、一直状态良好,待14天后灌流小鼠切片、玻片扫描仪成像。起初以为小鼠无感染症状、病毒可能被免疫清除,实际结果如图5所示,低毒HSV标记效果很好,注射VTA标记到的脑区有中脑导水管周围灰质(periaqueductal gray,PAG),中脑深部网状核(deep mesencephalic nucleus,DpMe),外侧下丘脑(lateral hypothalamic area,LH),外侧缰核(lateral habenular nucleus,LHb),伏隔核(Nucleus accumbens,NAc),腹侧苍白球(ventral pallidum,VP)等,而且标记的神经元胞体和轴/树突纤维都清晰可见,很多长的投射纤维都被高亮标记。
实施例6:
低毒单纯疱疹病毒高效示踪前嗅核神经网络
按常规操作,精确量取200nl实施例2制备的H129Δγ34.5-hUbC-tdTomato-WPRE(病毒滴度是2×109PFU/ml)立体定位注射C57BL/6小鼠大脑前嗅核(AON)脑区,小鼠无感染症状,待感染14天后麻醉动物,分别用0.9%(V/V)生理盐水灌流,然后用4%(V/V)多聚甲醛灌流;取出脑组织浸泡于4%(V/V)多聚甲醛液中,然后将脑组织先置于20%(V/V)蔗糖溶液中1天,接着置于30%(V/V)蔗糖溶液中2天;切片前将脑组织底部切平,置于底座上包埋冰冻1小时后切片;挑取脑片使用荧光显微镜观察。
低毒HSV注射前嗅核,也获得了理想的示踪标记效果,待14天小鼠不会死、一直状态良好。低毒HSV从前嗅核示踪标记到的脑区有外侧中膈核LSD,海马大片区域,后内侧皮质杏仁核PMCo,梨状皮层,顶盖脊髓束和大脑皮层的部分脑区等(见图6),荧光标记强、神经元结构细节很清晰。
实施例7:
低毒单纯疱疹病毒长时程(1月)、高效示踪VTA神经网络
基于低毒HSV中枢标记动物两周基本无症状及良好的标记效果,我们测试了更长的标记时间。按常规操作,精确量取200nl H129Δγ34.5-hUbC-tdTomato-WPRE(病毒滴度是2×109PFU/ml)立体定位注射C57BL/6小鼠大脑腹侧被盖区(VTA),待感染32天后麻醉动物,分别用0.9%(V/V)生理盐水灌流,然后用4%(V/V)多聚甲醛灌流;脊髓剥离步骤:剪开小鼠颈背部皮肤,暴露脊椎骨;剔除脊椎骨外围肌肉,使用咬骨钳咬住最外一节椎骨,缓慢拉出;重复第二步,去除所有椎骨,取出脊髓。取出的脑组织浸泡于4%(V/V)多聚甲醛液中,然后将脑组织先置于20%(V/V)蔗糖溶液中1天,接着置于30%(V/V)蔗糖溶液中2天;切片前将脑组织底部切平,置于底座上包埋冰冻1小时后切片;挑取脑片使用荧光显微镜观察。
结果如图7所示,低毒HSV注射VTA一个月,小鼠状态良好、未表现感染症状;出乎意料的长达1个月低毒HSVLT并没有被清除,而且获得了更好的标记效果,基地脑区、丘脑、海马、脑干很多脑区被高亮标记,荧光信号很强没有衰减(见图7中A)。另外由延髓外延至外周的脊髓都检测到高亮标记的神经元,在颈段、胸段脊髓也发现标记到的神经元(见图7中B)。从以上结果可以看出低毒HSVLT非常适合用于长时程神经环路示踪解析,较野生型H129表达荧光信号随时间逐渐衰减相比,低毒HSVLT展现出了长时程、高表达的标记特性优势,为开发应用于功能环路解析的HSV工具病毒打下了坚实基础。
实施例8:
低毒单纯疱疹病毒长时程(2月)、高效示踪M1神经网络
我们将低毒HSVLT注射小鼠初级运动皮层(M1)测试了更长的标记时间,长达2个月。按常规操作,精确量取200nl H129Δγ34.5-hUbC-tdTomato-WPRE工具病毒(病毒滴度是2×109PFU/ml),立体定位注射C57BL/6小鼠大脑初级运动皮层(M1),连续观察感染动物症状、待感染2个月后麻醉动物,分别用0.9%(V/V)生理盐水灌流,然后用4%(V/V)多聚甲醛灌流;取出脑组织浸泡于4%(V/V)多聚甲醛液中,然后将脑组织先置于20%(V/V)蔗糖溶液中1天,接着置于30%(V/V)蔗糖溶液中2天;切片前将脑组织底部切平,置于底座上包埋冰冻1小时后切片;挑取脑片使用荧光显微镜观察。
低毒HSVLT-tdT注射初级运动皮层(M1)两个月,小鼠一直状态良好、无感染症状;标记结果如图8所示,感染2个月低毒HSVLT也没有被清除,而且标记效果很好,高亮标记到的脑区有对侧M1、初级感觉皮层S1、尾状核CPu、黑质SN等,荧光信号强没有衰减。综上结合低毒HSVLT-tdT长时程标记VTA1个月的结果,HSVLT标记初级运动皮层两个月更进一步展示出了其低毒性、长时程高表达的标记特性。
实施例9:
低毒单纯疱疹病毒活体标记毒性测试
选取上述低毒HSVLT(H129Δγ34.5-hUbC-tdTomato-WPRE)200nl注射小鼠VTA脑区32天的脑片,以及表达tdTomato的野生型H129注射VTA脑区3天的脑片(野生型H129注射小鼠一般3-5天会死亡),用Caspase-3单抗做免疫组化进行神经元细胞凋亡检测,Caspase-3是细胞凋亡过程中最主要的终末剪切酶。
免疫组化步骤如下:①脑片用1×PBS(pH7.4)清洗3次,每次5min;②10%羊血清(1×PBS,0.3%triton)孵育脑片(1h,室温,150ul/孔);③吸去羊血清(不用清洗),加Caspase-3鼠单抗(一抗)(1×PBS,0.3%triton稀释1000倍),120ul/孔,孵育。4度放置过夜;④吸去一抗,1×PBS洗4次,每次5min;⑤加入二抗,羊抗鼠FITC(1×PBS,0.3%triton稀释400倍),120ul/孔,37度孵育1h;⑥吸去二抗,1×PBS洗3次,每次5min;⑦加入DAPI(1×PBS稀释5000倍),室温避光放置7-10min;⑧吸去DPPI,1×PBS洗3次,每次5min;⑨将脑片贴在防脱载玻片上,甘油(70%)封片,盖玻片边缘涂指甲油;⑩荧光显微镜观察脑片,并成像。
组化结果如图9所示,野生型H129-tdTomato标记的脑片中表达红色荧光的神经元部位检测到大量Caspase-3阳性的绿色信号(见图9的a1,a2),说明野生型H129感染3天的神经元产生炎症反应、诱发细胞凋亡;然而低毒H129LT即使感染32天的多个脑区均未检测到Caspase-3阳性绿色信号(见图9的b1,b2),而红色荧光信号很亮,结果很好地说明HS VLT(H129Δγ34.5-hUbC-tdTomato-WPRE)显著减毒,对中枢感染的动物以及标记到的神经元毒性很小,不会引起神经元细胞凋亡,这为开发表达功能探针用于神经环路功能活动解析的低毒HSV工具病毒打下了坚实的基础。
实施例10:
无外源启动子的重组低毒单纯疱疹病毒的构建及制备
(1)双敲除γ34.5基因、外源基因表达盒不含外源启动子的重组靶向载体构建
以pcDNA3.1-CMV-EGFP-WPRE-PA质粒为模板,设计引物直接克隆EGFP-WPRE-PA片段,不包含CMV启动子,所用引物序列为EGFPWPA-F:5'CCACCGGTGCCACCATGGTGAGCAAGGGCGAGGAGCTG 3'(SEQ ID NO.20);EGFPWPA-R:5'CGGGATCCCCATAGAGCCCACCGCATCCCCAG 3'(SEQ ID NO.21);后续将切胶回收纯化的EGFP-WPRE-PA大片段经Age I、BamH I酶切后连入双敲除γ34.5基因的重组质粒pH129Δγ34.5,获得最终的无外源启动子的表达EGFP靶向载体pH129Δγ34.5-EGFP-WPRE-PA(见图1C),经酶切、测序证明靶向载体构建正确。
(2)无外源启动子的重组低毒单纯疱疹病毒的重组制备
①病毒的重组:抽提靶向载体pH129Δγ34.5-EGFP-WPRE-PA,采用脂质体转染的方法转染293T细胞,6小时后更换含2%FBS的维持培养基并加入单纯疱疹病毒H129株进行感染;在不同时间观察荧光的表达和细胞病变情况,待细胞全部病变后收集细胞培养基上清置于-80℃冰箱。
②病毒的纯化:将收集的病毒上清经三次反复冻融、6500g离心10分钟去净细胞碎片,吸取10μl病毒上清感染Vero细胞,1天后观察感染细胞有无荧光表达来确定新型病毒是否重组成功;后期取重组成功的病毒上清连续10倍梯度稀释后感染Vero细胞,吸附1小时后铺琼脂(含5%胎牛血清的DMEM培养基和2%琼脂1:1混合)。48-72hr后待病毒斑形成后,在倒置荧光显微镜下进行挑斑;新型重组病毒经过6轮左右的挑斑纯化将野生型病毒去除,获得纯化的新型重组病毒H129Δγ34.5-EGFP-WPRE。无外源启动子的低毒单纯疱疹病毒的重组、挑斑及纯化后感染细胞荧光表达情况如图10所示,可见EGFP基因直接克隆于敲除的γ34.5上游同源臂(UHA)之后能得到有效表达,虽然荧光亮度明显弱于上述制备的含外源泛素启动子的H129Δγ34.5-hUbC-EGFP-WPRE-PA。以上结果说明γ34.5启动子就位于长527bp的UHA之中,γ34.5上游同源臂含有的启动子序列和重组引入的同向的外源启动子一起将显著增强外源基因的表达强度和效力。
序列表
<110> 中国科学院武汉物理与数学研究所
<120> 低毒单纯疱疹病毒系统及其构建方法和应用
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9980
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttagcc cgggcccccc gcgggcgggg cggcgcgcaa aaaaggcggc 960
cggcggcccg ggcggcgggc gcgcgcacgg cgggcgttgg gggcggggcc gcgggagcgg 1020
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1080
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1140
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1200
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1260
ccagacccca aaaacgggcc ccccccgaaa cacacccccc gggggtcgcg cgcggccctt 1320
taaagcgcgg cggcgcagcc cgggcccccc gcggctagcg agttagacag gcaagcacta 1380
ctcgcctctg cacgcacatg cttgcctgtc aaactctacc accccggcac gctctctgtc 1440
tccaccggtc catcgatggg ttaacggatc cactagtcca gtgtggtgga attcgcgccg 1500
ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga 1560
agggcgcagg agcgttcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc 1620
ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg 1680
tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct 1740
tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga 1800
cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt 1860
tgtggatcgc tgtgatcgtc acttggtgag ttgcgggctg ctgggctggc cggggctttc 1920
gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt 1980
ctgggtccgc gagcaaggtt gccctgaact gggggttggg gggagcgcac aaaatggcgg 2040
ctgttcccga gtcttgaatg gaagacgctt gtaaggcggg ctgtgaggtc gttgaaacaa 2100
ggtggggggc atggtgggcg gcaagaaccc aaggtcttga ggccttcgct aatgcgggaa 2160
agctcttatt cgggtgagat gggctggggc accatctggg gaccctgacg tgaagtttgt 2220
cactgactgg agaactcggg tttgtcgtct ggttgcgggg gcggcagtta tgcggtgccg 2280
ttgggcagtg cacccgtacc tttgggagcg cgcgcctcgt cgtgtcgtga cgtcacccgt 2340
tctgttggct tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct 2400
ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga 2460
cctctggtga ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta 2520
tcttcttaag tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt 2580
ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca 2640
gtgttagact agtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacgg 2700
taccatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat 2760
ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta 2820
cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg 2880
ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga 2940
catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa 3000
cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct 3060
gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa 3120
gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa 3180
gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa 3240
gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa 3300
gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga 3360
gggccgccac cacctgttcc tggggcatgg caccggcagc accggcagcg gcagctccgg 3420
caccgcctcc tccgaggaca acaacatggc cgtcatcaaa gagttcatgc gcttcaaggt 3480
gcgcatggag ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg 3540
cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt 3600
cgcctgggac atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc 3660
cgccgacatc cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt 3720
gatgaacttc gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg 3780
cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat 3840
gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt 3900
gctgaagggc gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga 3960
gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga 4020
caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg 4080
ctccgagggc cgccaccacc tgttcctgta cggcatggac gagctgtaca agtaatctag 4140
aaatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 4200
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 4260
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 4320
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 4380
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 4440
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 4500
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 4560
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 4620
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 4680
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc agggcccgtt 4740
taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 4800
tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 4860
gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 4920
caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 4980
tctatggcct gcaggaactt gcaagaggcc ttgttccgct tcccggtatg gtaattagaa 5040
actcattaat gggcggcccc ggccgccctt cccgcttccg gcaattcccg cggcccttaa 5100
tgggcaaccc cggtattccc cgcctcccgc gccgcgcgta accactcccc tggggttccg 5160
ggttatgcta attgcttttt tggcggaaca cacggcccct cgcgcattgg cccgcgggtc 5220
gctcaatgaa cccgcattgg tcccctgggg ttccgggtat ggtaatgagt ttcttcggga 5280
aggcgggaag ccccggggcg ccgacgcagg ccaagcccct gttgcgtcgg cgggaggggc 5340
atgctaatgg ggttctttgg gggacaccgg gttggtcccc caaatcgggg gccgggccgt 5400
gcatgctaat gatattcttt gggggcgccg ggttggtccc cggggacggg gccgccccgc 5460
ggtgggcctg cctcccctgg gacgcgcggc cattggggga atcgtcactg ccgccccttt 5520
ggggagggga aaggcgtggg gttctagagg gcccgtttaa acccgctgat cagcctcgac 5580
tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5640
ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5700
gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5760
ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 5820
aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 5880
gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 5940
tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 6000
tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 6060
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 6120
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 6180
ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 6240
aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 6300
ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 6360
aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 6420
agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 6480
ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 6540
gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 6600
ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 6660
caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 6720
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 6780
tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 6840
gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 6900
acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 6960
ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 7020
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 7080
ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 7140
cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 7200
gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 7260
tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 7320
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 7380
cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 7440
cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 7500
aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct 7560
tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 7620
gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 7680
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 7740
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 7800
ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 7860
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 7920
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 7980
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 8040
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 8100
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 8160
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 8220
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 8280
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 8340
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 8400
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 8460
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 8520
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 8580
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 8640
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 8700
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 8760
gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 8820
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 8880
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 8940
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9000
gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9060
tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9120
cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9180
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9240
gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9300
caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 9360
gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 9420
ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 9480
tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 9540
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 9600
tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 9660
cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 9720
ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 9780
aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 9840
tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 9900
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 9960
gaaaagtgcc acctgacgtc 9980
<210> 2
<211> 1007
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggcccgcc gccgccgccg ccatcgcggc ccccgccgcc cccggccgcc cgggcccacg 60
ggcgcggtcc caaccgcaca gtcccaggta acctccacgc ccaactcgga acccgtggtc 120
aggagcgcgc ccgcggccgc cccgccgccg ccccccgccg gtgggccccc gccttcttgt 180
tcgctgctgc tgcgccagtg gctccacgtt cccgagtccg cgtccgacga cgacgatgac 240
gacgactggc cggacagccc cccgcccgag ccggcgccag acgcccggcc caccgccgcc 300
gccccccgcc cccggtcccc accgcccggc gcgggcccgg ggggcggggc taacccctcc 360
caccccccct cacgcccctt ccgccttccg ccgcgcctcg ccctccgcct gcgcgtcacc 420
gcagagcacc tggcgcgcct gcgacgcgcg ggcggggagg gggcgccgga gccccccgcg 480
acccccgcga cccccgcgac ccccgcgcgg gtgcgcttct cgccccacgt ccgggtgcgc 540
cacctggtgg tctgggcctc ggccgcccgc ctggcgcgcc gcggctcgtg ggcccgcgag 600
cgggccgacc gggctcggtt ccggcgccgg gtggcggagg ccgaggcggt catcgggccg 660
tgcctggggc ccgaggcccg tgcccgggcc ctggcccgcg gagccggccc ggcgaactcg 720
gtctaacgtt acacccgagg cggcctgggt cttccgcgga gctcccggga gctccgcacc 780
aagccgctct ccggagagac gatggcagga gccgcgcata tatacgctgg gagccggccc 840
gcccccaagg cgggcccgcc ctcggagggc gggactggcc aatcggcggc cgccagcgcg 900
gcggggcccg gccaaccagc gtttgccgag tcttcggggc ccggcccact gggcggtaac 960
tcccgcccag tgggccgggc cgcccacttc ccggtatggt aattaaa 1007
<210> 3
<211> 1431
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60
ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120
ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180
atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240
cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300
gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360
tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420
accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480
gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540
atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600
gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660
cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc 720
gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc 780
atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 840
tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 900
tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 960
gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1020
aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 1080
ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 1140
aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 1200
aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 1260
aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 1320
aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 1380
gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaagta a 1431
<210> 4
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 5
<211> 8069
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttagcc cgggcccccc gcgggcgggg cggcgcgcaa aaaaggcggc 960
cggcggcccg ggcggcgggc gcgcgcacgg cgggcgttgg gggcggggcc gcgggagcgg 1020
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1080
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1140
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1200
ggggaggagc ggggggagga gcggggggag gagcgggggg aggagcgggg ggaggagcgg 1260
ccagacccca aaaacgggcc ccccccgaaa cacacccccc gggggtcgcg cgcggccctt 1320
taaagcgcgg cggcgcagcc cgggcccccc gcggctagcg agttagacag gcaagcacta 1380
ctcgcctctg cacgcacatg cttgcctgtc aaactctacc accccggcac gctctctgtc 1440
tccaccggtg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 1500
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 1560
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 1620
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 1680
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1740
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 1800
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1860
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 1920
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 1980
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2040
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2100
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2160
gagctgtaca agtaatctag aaatcaacct ctggattaca aaatttgtga aagattgact 2220
ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 2280
tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 2340
ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 2400
tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 2460
actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 2520
tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 2580
tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 2640
tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 2700
ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 2760
gcctccccgc agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc 2820
agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 2880
ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 2940
ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 3000
atgctgggga tgcggtgggc tctatgggga tccactagtc cagtgtggtg gaattcattt 3060
aaatccttaa ttaaggcctg caggaacttg caagaggcct tgttccgctt cccggtatgg 3120
taattagaaa ctcattaatg ggcggccccg gccgcccttc ccgcttccgg caattcccgc 3180
ggcccttaat gggcaacccc ggtattcccc gcctcccgcg ccgcgcgtaa ccactcccct 3240
ggggttccgg gttatgctaa ttgctttttt ggcggaacac acggcccctc gcgcattggc 3300
ccgcgggtcg ctcaatgaac ccgcattggt cccctggggt tccgggtatg gtaatgagtt 3360
tcttcgggaa ggcgggaagc cccggggcgc cgacgcaggc caagcccctg ttgcgtcggc 3420
gggaggggca tgctaatggg gttctttggg ggacaccggg ttggtccccc aaatcggggg 3480
ccgggccgtg catgctaatg atattctttg ggggcgccgg gttggtcccc ggggacgggg 3540
ccgccccgcg gtgggcctgc ctcccctggg acgcgcggcc attgggggaa tcgtcactgc 3600
cgcccctttg gggaggggaa aggcgtgggg ttctagaggg cccgtttaaa cccgctgatc 3660
agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 3720
cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 3780
gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 3840
ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 3900
ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 3960
aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 4020
gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 4080
agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 4140
caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 4200
tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 4260
aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 4320
ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 4380
gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 4440
atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 4500
agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 4560
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 4620
tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga 4680
ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc 4740
ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac 4800
gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 4860
atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 4920
gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 4980
tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 5040
agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 5100
cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 5160
gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 5220
gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 5280
gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 5340
ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 5400
tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 5460
gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 5520
cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 5580
tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca 5640
ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 5700
tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag 5760
cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 5820
cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 5880
cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 5940
gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 6000
gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 6060
cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 6120
tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 6180
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 6240
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6300
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6360
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6420
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6480
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 6540
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 6600
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6660
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6720
tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 6780
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 6840
ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 6900
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 6960
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 7020
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 7080
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 7140
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 7200
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 7260
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 7320
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 7380
ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 7440
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 7500
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 7560
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 7620
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 7680
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 7740
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 7800
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 7860
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 7920
gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 7980
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 8040
catttccccg aaaagtgcca cctgacgtc 8069
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cccaagctta gcccgggccc cccgcgggc 29
<210> 7
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgggatccgt taacccatcg atggaccggt ggagacagag agcgtgccgg 50
<210> 8
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccggaattca tttaaatcct taattaaggc ctgcaggaac ttgcaagagg ccttgttc 58
<210> 9
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gctctagaac cccacgcctt tcccctcc 28
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctagctagcg cgccgggttt tggcgcctcc cgcg 34
<210> 11
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cggggtaccg tctaacaaaa aagccaaaaa cggc 34
<210> 12
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgctctagaa atcaacctct ggattacaaa atttg 35
<210> 13
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaagggccct gcggggaggc ggcccaaagg gagatc 36
<210> 14
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ataggtacca tggtgagcaa gggcgaggag g 31
<210> 15
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgctctagat tacttgtaca gctcgtccat gccg 34
<210> 16
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agtccagtgt ggtggaattc gcgccgggtt ttggcgcctc 40
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctcttgcaag ttcctgcagg ccatagagcc caccgcatcc 40
<210> 18
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggcccgcc gccgccgccg ccatcgcggc ccccgccgcc cccgg 45
<210> 19
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ttagaccgag ttcgccgggc cggctccgcg ggccagggcc cgggc 45
<210> 20
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccaccggtgc caccatggtg agcaagggcg aggagctg 38
<210> 21
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cgggatcccc atagagccca ccgcatcccc ag 32
Claims (9)
1.一种重组低毒单纯疱疹病毒,所述的低毒HSV通过将HSV-1 H129基因组中双拷贝的神经毒力因子γ34.5基因完全敲除,将完整的外源基因表达盒插入敲除的γ34.5基因座位中得到。
2.根据权利要求1所述的重组靶向载体,所述的完整的荧光基因表达盒包括:hUbC启动子、荧光基因和WPRE转录增强元件等。
3.一种制备携带红色荧光基因(SEQ ID NO.3)的低毒单纯疱疹病毒所用靶向载体pH129Δγ34.5-hUbC-tdTomato-WPRE-PA,序列为SEQ ID NO.1所示。
4.一种制备携带绿色荧光基因(SEQ ID NO.4)前无外源启动子的低毒单纯疱疹病毒所用靶向载体pH129Δγ34.5-EGFP-WPRE-PA,序列为SEQ ID NO.5所示。
5.利用权利要求3所述的靶向载体重组制备的低毒单纯疱疹病毒系统(命名为H129LT(low-toxic))。
6.利用权利要求3所述的靶向载体重组制备的低毒单纯疱疹病毒系统,其特征在于,所述外源基因表达盒包括一个启动子驱动外源基因表达,启动子可以是hUbC启动子、CMV启动子、CAG启动子、EF1α启动子、pTH启动子、pChAT启动子等。
7.利用权利要求3所述的靶向载体重组制备的低毒单纯疱疹病毒系统,其特征在于,所述外源基因表达盒表达一个由基因序列(SEQ ID NO.3)编码的红色荧光蛋白或由基因序列(SEQ ID NO.4)编码的绿色荧光蛋白,或其变体,或者其它任何目的外源基因;
因此可以用其它任何目的基因替代本发明用的荧光基因构建定制表达目的基因的低毒单纯疱疹病毒。
8.权利要求1所述的重组靶向载体或权利要求5所述的低毒单纯疱疹病毒系统的应用;所述的应用包括:神经环路标记示踪、大容量基因转导载体、神经系统靶向基因治疗、病毒复制与致病机理分析、动物感染模型建立、抗病毒药物筛选、溶瘤治疗等。
9.利用权利要求4所述的靶向载体重组制备的低毒单纯疱疹病毒及其应用,可用于分析γ34.5基因上游的启动子位置、组成、表达调控活性及在病毒复制过程中的作用等。
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CN110305878A (zh) * | 2019-04-16 | 2019-10-08 | 华中农业大学 | 一种牛分枝杆菌卡介苗低黏附力及低侵袭力突变株b2909 |
CN112063658A (zh) * | 2020-09-22 | 2020-12-11 | 昆明医科大学第二附属医院 | 一种基于hsv-1的同源重组载体及其靶点序列和应用 |
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CN114164165A (zh) * | 2021-12-14 | 2022-03-11 | 中国科学院大连化学物理研究所 | 一种微流控芯片在构建疱疹性脑炎模型中的应用 |
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WO2023065502A1 (zh) * | 2021-10-22 | 2023-04-27 | 中国科学院深圳先进技术研究院 | 重组单纯疱疹病毒的亲和筛选细胞系及其构建方法和应用 |
WO2023087464A1 (zh) * | 2021-11-16 | 2023-05-25 | 中国科学院深圳先进技术研究院 | 基于中国hsv临床分离株的溶瘤病毒及其构建方法和应用 |
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WO2023087464A1 (zh) * | 2021-11-16 | 2023-05-25 | 中国科学院深圳先进技术研究院 | 基于中国hsv临床分离株的溶瘤病毒及其构建方法和应用 |
CN114164165A (zh) * | 2021-12-14 | 2022-03-11 | 中国科学院大连化学物理研究所 | 一种微流控芯片在构建疱疹性脑炎模型中的应用 |
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