CN116334010B - 一种重组单纯疱疹病毒及其制备方法和应用 - Google Patents
一种重组单纯疱疹病毒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,公开了一种重组单纯疱疹病毒及其制备方法和应用,所述重组单纯疱疹病毒包括载体和用于降低DPD基因表达水平的核苷酸序列;载体为缺失编码ICP47基因并含有CD基因的单纯疱疹病毒,所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上缺失编码ICP47的基因的位置;该重组单纯疱疹病毒将干扰DPD的shRNA插入到HSV‑CD中,在干扰胶质瘤细胞代谢的同时提高CD/5‑FC对胶质瘤细胞的杀伤能力。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种重组单纯疱疹病毒及其制备方法和应用。
背景技术
溶瘤病毒已经在多种临床前和临床研究中用于癌症治疗,溶瘤病毒是通过删除病毒基因组上的特定基因从而赋予溶瘤病毒选择性在肿瘤细胞内复制扩增能力,获得肿瘤杀伤效果,同时改构后的溶瘤病毒在正常细胞中无法复制,因而具备较高的安全性;I型单纯疱疹病毒(herpes simplex virus I,HSV-1)是目前在临床使用最广泛的溶瘤病毒之一。
目前有利用基因工程手段将野生型HSV-1进行改造,删除特定基因后作为基因表达载体应用于人体基因治疗的研究,在溶瘤的同时表达治疗基因协同溶瘤病毒治疗;在改造野生型HSV-1的基础上插入了2个表达框架,一个是肿瘤细胞中能够表达胞嘧啶脱氨酶(cytosine deaminase,CD),CD可以将外源的无毒5-FC(前药5-氟胞嘧啶)在细胞内转化为化疗药5-FU(5-氟尿嘧啶),干扰肿瘤细胞DNA合成;另外一个是插入了干扰TSPO基因的shRNA,降低其表达,经过三重改造后构建HSV-CD-shTSPO溶瘤病毒。
上述重组的单纯疱疹病毒能够有效降低脑胶质瘤生长速度,可以为HSV-1溶瘤病毒提供充分的时间以杀伤脑胶质瘤细胞,提高了脑胶质瘤的杀伤效率,而且使用后副反应小;但该技术中插入的干扰TSPO基因的shRNA,虽然发挥干扰胶质瘤细胞代谢的作用,但是与病毒中插入的CD基因没有直接关系,其不能直接增效CD/5-FC的杀伤功能。
发明内容
针对上述问题,本发明的第一个目的是提供一种重组单纯疱疹病毒,该重组单纯疱疹病毒将干扰DPD的shRNA插入到HSV-CD中,在干扰胶质瘤细胞代谢的同时提高CD/5-FC对胶质瘤细胞的杀伤能力。
本发明的第二个目的是提供上述一种重组单纯疱疹病毒的制备方法。
本发明的第三个目的是提供一种重组单纯疱疹病毒在制备用于治疗脑胶质瘤的药物中的应用。
本发明所采用的第一个技术方案是:一种重组单纯疱疹病毒,所述重组单纯疱疹病毒包括载体和用于降低DPD基因表达水平的核苷酸序列;
所述载体为缺失编码ICP47基因并含有CD基因的单纯疱疹病毒,所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上缺失编码ICP47的基因的位置;
所述用于降低DPD基因表达水平的核苷酸序列如SEQ ID No:1、SEQ ID No:2、SEQID No:3或SEQ ID No:4所示。
优选地,所述载体中CD基因的插入位点为缺失编码ICP34.5基因的位点。
优选地,所述DPD基因具有如SEQ ID No:5所示的核苷酸序列。
优选地,所述CD基因具有如SEQ ID No:6所示的核苷酸序列。
优选地,所述载体通过以下方式获得:
将单纯疱疹病毒中编码ICP34.5的基因敲除,在敲除编码ICP34.5的基因的位置上插入CD基因,从而获得含有CD基因的单纯疱疹病毒;
将含有CD基因的单纯疱疹病毒中编码ICP47的基因敲除,从而获得载体。
本发明所采用的第二个技术方案是:一种制备第一个技术方案中所述的重组单纯疱疹病毒的方法,包括以下步骤:
将含有CD基因的单纯疱疹病毒中编码ICP47的基因敲除,从而获得载体;在载体上插入用于降低DPD基因表达水平的核苷酸序列;所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上敲除编码ICP47的基因的位置;
所述用于降低DPD基因表达水平的核苷酸序列如SEQ ID No:1、SEQ ID No:2、SEQID No:3或SEQ ID No:4所示。
优选地,所述含有CD基因的单纯疱疹病毒通过以下方式获得:
将单纯疱疹病毒中编码ICP34.5的基因敲除,在敲除编码ICP34.5的基因的位置上插入CD基因,从而获得含有CD基因的单纯疱疹病毒。
优选地,所述的制备重组单纯疱疹病毒的方法还包括:
合成CD基因,并用HindⅢ和XhoⅠ将CD基因克隆到pSP72△ICP34.5CMVWPRE载体中得到pSP72△ICP34.5CMVCDWPRE;pSP72△ICP34.5CMVCDWPRE与HSV-1△ICP34.5CMVEGFP共同转染BHK细胞,不表达绿色荧光的病毒斑表示原病毒中的基因EGFP由CD基因重组而替换,纯化及生长HSV-1△ICP34.5CMVCDWPRE病毒载体,提取该载体全长DNA,即获得含有CD基因的单纯疱疹病毒。
本发明所采用的第三个技术方案是:根据第一个技术方案中所述的重组单纯疱疹病毒或由第二个技术方案中所述的方法制备得到的重组单纯疱疹病毒在制备用于治疗脑胶质瘤的药物中的应用。
上述技术方案的有益效果:
(1)本发明将干扰DPD的shRNA插入到HSV-CD中,构建HSV-CD-shDPD溶瘤病毒(即重组单纯疱疹病毒),重组单纯疱疹病毒有效降低胶质瘤代谢、侵袭迁移,同时提高插入的CD基因对前药5-FC的利用率,增加CD基因转化的5-FU半衰期及对胶质瘤细胞内的化疗效率,即同时提高对外源药物的利用率,增加化疗药物半衰期以及对胶质瘤细胞内的化疗效率。
附图说明
图1为脑胶质瘤不同病理级别与DPD表达的关系图;
图2为原发脑胶质瘤中DPD表达与预后的关系图;
图3为复发脑胶质瘤中DPD表达与预后的关系图;
图4为免疫组化检测脑胶质瘤病理组织中DPD表达的示意图,其中Low grade为低级别,High grade为高级别;
图5为不同干扰DPD的shRNA的干扰效率图;
图6为胶质瘤细胞U251中干扰DPD表达抑制细胞侵袭和迁移能力的效果图;
图7为干扰胶质瘤细胞中DPD表达,并增强了5-FU杀伤细胞能力的效果图;
图8为sh-DPD-1#、sh-DPD-3#和sh-TSPO对5-FC的利用率的对比图。
具体实施方式
下面通过具体的实施例对本发明进一步说明,应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干变型和改进,这些也应视为属于本发明的保护范围。
DPD(二氢嘧啶脱氢酶)是5-FU(5-氟尿嘧啶)体内代谢的起始酶和限速酶,可将细胞中5-FU转化为无毒的二氢氟尿嘧啶,是CD(胞嘧啶脱氨酶)/5-FC系统的抑制酶;DPD基因(DPYD)不仅参与代谢过程还参与肿瘤EMT过程;本发明的发明人基于中国脑胶质瘤基因组图谱计划(CGGA)数据库进行生物信息学分析DPD(二氢嘧啶脱氢酶)表达与胶质瘤预后的关系,如图1所示,发现胶质瘤病理级别越高(Ⅱ、Ⅲ到Ⅳ级),DPD表达越高;如图2和图3所示,原发胶质瘤和复发胶质瘤中,DPD表达高,胶质瘤预后差;即DPD(二氢嘧啶脱氢酶)在脑胶质瘤病理组织中普遍高表达,并且病理级别越高,DPD表达越高;体外干扰胶质瘤细胞中DPD表达,不仅抑制胶质瘤细胞侵袭迁移,同时还能增强5-FU杀伤胶质瘤细胞能力。
根据以上生物信息学分析结果,免疫组化分析了DPD在低级别(Ⅱ)和高级别(Ⅲ或Ⅳ级)病理组织中表达,如图4所示,发现DPD在高级别比低级别表达高,进一步验证了上述分析结果。
基于以上分析,本发明一方面提供了一种重组单纯疱疹病毒,所述重组单纯疱疹病毒包括载体和用于降低DPD基因表达水平的核苷酸序列(即干扰DPD的shRNA);所述载体为缺失编码ICP47基因并含有CD基因的单纯疱疹病毒,所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上缺失编码ICP47的基因的位置;
其中,所述用于降低DPD基因表达水平的核苷酸序列如SEQ ID No:1、SEQ ID No:2、SEQ ID No:3或SEQ ID No:4所示;优选如SEQ ID No:1或SEQ ID No:3所示的核苷酸序列;缺失编码ICP47基因并含有CD基因的单纯疱疹病毒中CD基因的插入位点为缺失编码ICP34.5基因的位点上。
在本发明中,所述编码ICP34.5的基因和编码ICP47的基因均为本领域技术人员所公知,并且也能够通过登录相关的数据库查到,例如,可以通过登录GenBank数据库查询到相关的核苷酸序列,这些均是本领域技术人员具有的常规技术手段,本发明在此不再赘述。
所述DPD基因具有如SEQ ID No:5所示的核苷酸序列。
所述CD基因具有如SEQ ID No:6所示的核苷酸序列;
前药5-氟胞嘧啶(5-FC)对细胞无作用;由5-FC脱氨基转化得到的5-FU对正常细胞和肿瘤细胞都有杀伤作用,这一过程可由胞嘧啶脱氨酶(CD)进行催化,而人体中缺少此类脱氨酶;胞嘧啶脱氨酶(CD基因的表达产物),能将原本无毒的前体药物5-FC转化成具有细胞毒性的化疗药物5-FU。
在本发明中,对所述单纯疱疹病毒的种类没有特别的限定,可以为本领域的常规选择,但是为了更好地实现稳定表达细胞因子的目的,所述单纯疱疹病毒优选为I型单纯疱疹病毒;本发明对所述单纯疱疹病毒的来源也没有特别的限定,可以通过常规的商购获得,也可以通过实验室自行分离获得。
本发明另一方面还提供了一种重组单纯疱疹病毒的制备方法,包括:将含有CD基因的单纯疱疹病毒中编码ICP47的基因敲除,从而获得载体;在载体上插入用于降低DPD基因表达水平的核苷酸序列;所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上敲除编码ICP47的基因的位置。
其中,含有CD基因的单纯疱疹病毒通过以下方式获得:
将单纯疱疹病毒中编码ICP34.5的基因敲除,在敲除编码ICP34.5的基因的位置上插入CD基因,从而获得含有CD基因的单纯疱疹病毒。
编码ICP34.5、ICP47的基因的敲除可以采用本领域常规的各种方法,本发明对此并没有特别的限制,例如,通过同源重组的方式,通过定点敲除的方式;或者,还可以通过CRISPY的方式定点敲除。在了解了本发明的发明目的以及本发明使用的病毒载体的前提下,本领域技术人员根据其掌握的常规技术手段能够实现对如上基因的敲除。
用于降低DPD基因表达水平的核苷酸序列以及CD基因的插入也可以采用本领域常规的各种方法,所述插入的方式可以为直接在选定的插入位点插入所述目的序列,例如,可以通过CRISPY的方式插入,也可以通过同源重组的方式替换部分碱基序列从而插入所述目的序列。
重组单纯疱疹病毒的制备例如包括:
(1)合成CD基因,并用HindⅢ和XhoⅠ将CD基因克隆到pSP72△ICP34.5CMVWPRE载体(将包含有ICP34.5基因的HSV-1DNA片段插入质粒pSP72的BglII位点,编码ICP34.5基因的NotI的片段经由NotI限制内切后删除,得到psP72ΔICP34.5;由人类CMV病毒的IE启动子所控制,加上WPRE(Woodchuckhepatitispost-regulationElement)得到pSP72ΔICP34.5CMVWPRE载体)中得到pSP72△ICP34.5CMVCDWPRE;pSP72△ICP34.5CMVCDWPRE与HSV-1△ICP34.5CMVEGFP(从野生型的HSV-1中删除了ICP34.5基因,得到HSV-1△ICP34.5;表达绿色荧光蛋白的标记基因EGFP由人类CMV病毒的IE启动子所控制,共同克隆进HSV-1△ICP34.5,得到HSV-1△ICP34.5CMVEGFP)共同转染BHK细胞,不表达绿色荧光的病毒斑表示原病毒中的基因EGFP由CD基因重组而替换,纯化及生长HSV-1△ICP34.5CMVCDWPRE病毒载体,提取该载体全长DNA,即获得含有CD基因的单纯疱疹病毒;
(2)将SEQ ID No:1、SEQ ID No:2、SEQ ID No:3或SEQ ID No:4所示的核苷酸序列进行人工合成(人工合成由所委托的湖南亚大丰晖新材料有限公司完成),从而获得人工合成的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列或第四核苷酸序列;将含有CD基因的单纯疱疹病毒的ICP47基因敲除,并在敲除ICP47基因的位置插入上述人工合成的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列或第四核苷酸序列,从而获得重组单纯疱疹病毒;
(3)进行酶切验证,将重组单纯疱疹病毒进行测序验证(测序验证委托北京天一辉远生物科技有限公司完成),确认重组单纯疱疹病毒的阅读框正确,选出用于降低DPD基因表达水平的核苷酸序列正确插入到含有CD基因的单纯疱疹病毒中的病毒,作为重组单纯疱疹病毒。
本发明公开的重组单纯疱疹病毒能用于治疗脑胶质瘤的药物的制备。
以下将通过实施例对本发明进行详细描述。
实施例1
制备含有CD基因的单纯疱疹病毒:
按照申请号2004100064921,授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒(其基因序列的GenBank号:NC_001806)的ICP34.5基因敲除,并在HSV-1病毒的敲除ICP34.5基因的位置插入CD基因,从而获得含有CD基因的单纯疱疹病毒(即HSV-CD)。
实施例2
将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除(敲除方法同实施例1中的相同),并在敲除ICP47基因的位置插入用于降低DPD基因表达水平的如SEQ ID No:1所示(GCAATTTGCTACTGAGGTATT)的核苷酸序列(DPYD-1#);再与质粒载体构建重组病毒载体;构建成功的重组病毒载体在Vero细胞上于37℃、5%CO2条件下增殖,感染复数为0.1,收获后用0.65μm滤器去除细胞碎片,然后在13000rpm条件下高速离心纯化,得到滴度为1×108pfu/mL的病毒悬液,即获得重组单纯疱疹病毒HSV-CD-shDPD-1#(即sh-DPD-1#),用于后续的细胞实验。
实施例3
将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除(敲除方法同实施例1中的相同),并在敲除ICP47基因的位置插入用于降低DPD基因表达水平的如SEQ ID No:2所示(GCCGTATGATGTAGTGAATTT)的核苷酸序列(DPYD-2#);再与质粒载体构建重组病毒载体;构建成功的重组病毒载体在Vero细胞上于37℃、5%CO2条件下增殖,感染复数为0.1,收获后用0.65μm滤器去除细胞碎片,然后在13000rpm条件下高速离心纯化,得到滴度为1×108pfu/mL的病毒悬液,即获得重组单纯疱疹病毒HSV-CD-shDPD-2#(即sh-DPD-2#),用于后续的细胞实验。
实施例4
将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除(敲除方法同实施例1中的相同),并在敲除ICP47基因的位置插入用于降低DPD基因表达水平的如SEQ ID No:3所示(GCTATACAGTTTGATCCAGAA)的核苷酸序列(DPYD-3#);再与质粒载体构建重组病毒载体;构建成功的重组病毒载体在Vero细胞上于37℃、5%CO2条件下增殖,感染复数为0.1,收获后用0.65μm滤器去除细胞碎片,然后在13000rpm条件下高速离心纯化,得到滴度为1×108pfu/mL的病毒悬液,即获得重组单纯疱疹病毒HSV-CD-shDPD-3#(即sh-DPD-3#),用于后续的细胞实验。
实施例5
将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除(敲除方法同实施例1中的相同),并在敲除ICP47基因的位置插入用于降低DPD基因表达水平的如SEQ ID No:4所示(GCAAGTATAAGTTGTGCTTCC)的核苷酸序列(DPYD-4#);再与质粒载体构建重组病毒载体;构建成功的重组病毒载体在Vero细胞上于37℃、5%CO2条件下增殖,感染复数为0.1,收获后用0.65μm滤器去除细胞碎片,然后在13000rpm条件下高速离心纯化,得到滴度为1×108pfu/mL的病毒悬液,即获得重组单纯疱疹病毒HSV-CD-shDPD-4#(即sh-DPD-4#),用于后续的细胞实验。
对比例1
按照实施例2中的方法进行重组单纯疱疹病毒的构建,不同的是,仅将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除,未插入用于降低DPD基因表达水平的核苷酸序列,得到HSV-CD-shcon(即sh-con)。
对比例2
按照实施例2中的方法进行重组单纯疱疹病毒的构建,不同的是,将含有CD基因的单纯疱疹病毒的ICP47基因进行敲除后,并在敲除ICP47基因的位置插入用于降低TSPO基因表达水平的核苷酸序列,得到重组单纯疱疹病毒HSV-CD-sh-TSPO,该用于降低TSPO基因表达水平的核苷酸序列为:
TCACTCAACTACTGCGTATGTTCAAGAGACATACGCAGTAGTTGAGTGTTTTTT。
本发明中的DPYD-1#、DPYD-2#、DPYD-3#、DPYD-4#、CD基因和shTSPO是由湖南亚大丰晖新材料有限公司合成的;Vero细胞购自ATCC,货号为CCL-81。
测试例1
将上述实施例2(sh-DPD-1#)、实施例3(sh-DPD-2#)、实施例4(sh-DPD-3#)、实施例5(sh-DPD-4#)和对比例1(sh-con)中的重组单纯疱疹病毒分别转染至胶质瘤细胞U251中,转染过程(使用3000转染试剂,购自Thermo公司,方法参照说明书)包括:将胶质瘤细胞U251接种到24孔板中,37℃、5%CO2下增殖培养,贴壁后加入相应的实施例2-5和对比例1中构建的重组单纯疱疹病毒(感染复数MOI=0.01),分别感染胶质瘤细胞U251,37℃、5%CO2培养24h后收集细胞,Western blot检测sh-DPD-1#、sh-DPD-2#、sh-DPD-3#、sh-DPD-4#和sh-con转染后细胞中DPD蛋白的表达,从而获得用于降低DPD基因表达水平的不同核苷酸序列的干扰效率;其中,以未转染干扰DPD的shRNA的正常U251细胞(即sh-con)为对照,以肌动蛋白(Actin)为参比蛋白;检测结果如图5所示,由图5可知,DPYD-1#、DPYD-2#、DPYD-3#和DPYD-4#均能有效降低DPD基因的表达,其中DPYD-1#和DPYD-3#降低DPD基因表达的效果最好,即干扰DPD基因表达的效率较高。
测试例2
将上述实施例2(sh-DPD-1#)、实施例4(sh-DPD-3#)和对比例1(sh-con)中的重组单纯疱疹病毒分别转染至胶质瘤细胞U251中(所采用的转染方法同测试例1中的相同),将sh-DPD-1#、sh-DPD-3#和sh-con转染至胶质瘤细胞U251后,通过Transwell实验检测细胞侵袭能力和迁移能力;检测结果如图6所示,由图6可知,DPYD-1#和DPYD-3#(即干扰DPD的shRNA)表达后,胶质瘤细胞侵袭和迁移能力降低,而且DPYD-1#(即sh-DPD-1#)干扰DPD表达的效果要优于DPYD-3#(即sh-DPD-3#),干扰DPD表达的效果越好,抑制细胞侵袭和迁移的效果越明显。
DPYD-1#和DPYD-3#干扰DPD表达后,通过CCK8检测肿瘤细胞活性来检测5-FU杀伤细胞的能力,检测结果如图7所示,从图7中可知,未加入5-FU(即图7中的-5-FU)时,干扰DPD后,胶质瘤细胞活性下降;加入5-FU(即图7中的+5-FU)时,5-FU(5-FU的浓度为100umol/L)能有效杀伤胶质瘤细胞,并且干扰DPD表达的细胞中加入5-FU后胶质瘤细胞活性进一步下降,且DPYD-1#(即sh-DPD-1#)的效果优于DPYD-3#(即sh-DPD-3#)的效果,证明了干扰DPD表达后,胶质瘤细胞对5-FU更敏感,增加化疗药物半衰期以及对胶质瘤细胞内的化疗效率。
测试例3
将上述实施例2(sh-DPD-1#,即HSV-CD-shDPD-1#)、实施例4(sh-DPD-3#,即HSV-CD-shDPD-3#)和对比例2(HSV-CD-sh-TSPO)中的重组单纯疱疹病毒分别转染至胶质瘤细胞U251中(所采用的转染方法同测试例1中的相同),将sh-DPD-1#、sh-DPD-3#和HSV-CD-sh-TSPO转染至胶质瘤细胞U251后,DPYD-1#和DPYD-3#干扰DPD表达,通过CCK8检测肿瘤细胞活性来检测5-FU杀伤细胞的能力,检测结果如图8所示,从图8中可知,未加入5-FC(即图8中的-5-FC)时,干扰DPD后,胶质瘤细胞活性下降,而且干扰DPD后胶质瘤细胞的活性比干扰TSPO后胶质瘤细胞的活性更低;加入5-FC(即图8中的+5-FC)时,由胞嘧啶脱氨酶(CD)催化将5-FC脱氨基转化得到的5-FU,5-FU能有效杀伤胶质瘤细胞,并且相较于干扰TSPO表达的细胞中加入5-FC后胶质瘤细胞活性,本发明干扰DPD表达的细胞中加入5-FC后胶质瘤细胞活性进一步下降,证明了插入用于降低DPD基因表达水平的核苷酸序列能有效提高插入的CD基因对前药5-FC的利用率,即同时提高对外源药物的利用率。
上述测试例中胶质瘤细胞系U251购买自ATCC,货号为TX-1725。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (5)
1.一种重组单纯疱疹病毒,其特征在于,所述重组单纯疱疹病毒包括载体和用于降低DPD基因表达水平的核苷酸序列;
所述载体为缺失编码ICP47基因并含有CD基因的单纯疱疹病毒,所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上缺失编码ICP47的基因的位置;
所述用于降低DPD基因表达水平的核苷酸序列如SEQ ID No:1所示;
其中,所述载体通过以下方式获得:
将单纯疱疹病毒中编码ICP34.5的基因敲除,在敲除编码ICP34.5的基因的位置上插入CD基因,从而获得含有CD基因的单纯疱疹病毒;
将含有CD基因的单纯疱疹病毒中编码ICP47的基因敲除,从而获得载体。
2.根据权利要求1所述的重组单纯疱疹病毒,其特征在于,所述DPD基因具有如SEQ IDNo:5所示的核苷酸序列。
3.根据权利要求1所述的重组单纯疱疹病毒,其特征在于,所述CD基因具有如SEQ IDNo:6所示的核苷酸序列。
4.一种制备权利要求1-3中任意一项所述的重组单纯疱疹病毒的方法,其特征在于,包括以下步骤:
将单纯疱疹病毒中编码ICP34.5的基因敲除,在敲除编码ICP34.5的基因的位置上插入CD基因,从而获得含有CD基因的单纯疱疹病毒;
将含有CD基因的单纯疱疹病毒中编码ICP47的基因敲除,从而获得载体;在载体上插入用于降低DPD基因表达水平的核苷酸序列;所述用于降低DPD基因表达水平的核苷酸序列的插入位点为所述载体上敲除编码ICP47的基因的位置;
所述用于降低DPD基因表达水平的核苷酸序列如SEQ ID No:1所示。
5.根据权利要求1-3中任意一项所述的重组单纯疱疹病毒或由权利要求4中所述的方法制备得到的重组单纯疱疹病毒在制备用于治疗脑胶质瘤的药物中的应用。
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