RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed ...RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.展开更多
Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer v...Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.展开更多
利用单纯疱疹病毒I型(Herpes simplex virus type 1,HSV-1)标准株F细菌人工染色体(HSV-BAC)系统构建的HSV重组病毒由于存在tk基因缺陷,无法表达胸苷激酶,影响病毒潜伏/激活,故需对该基因进行修复。为此,本研究建立了tk1基因敲除的Vero...利用单纯疱疹病毒I型(Herpes simplex virus type 1,HSV-1)标准株F细菌人工染色体(HSV-BAC)系统构建的HSV重组病毒由于存在tk基因缺陷,无法表达胸苷激酶,影响病毒潜伏/激活,故需对该基因进行修复。为此,本研究建立了tk1基因敲除的Vero细胞株(tk1-Vero),并利用该细胞株建立了次黄嘌呤-氨基蝶呤-胸腺嘧啶脱氧核苷(Hypoxanthine-aminopurine-thymidine,HAT)选择性培养液筛选方法,对经同源重组方式完成tk基因修复的病毒进行筛选。首先,利用CRISPR/Cas9基因编辑技术敲除Vero细胞tk1基因,通过有限稀释法建立tk1-Vero细胞株,采用DNA测序和Western Blot对tk1-Vero细胞株进行鉴定;比较HSV(F)和tk-HSV(F)在tk1-Vero细胞和tk基因缺陷的人骨肉瘤143细胞中的增殖情况;在tk1-Vero细胞中,采用HAT选择性培养液筛选方法,对tk基因修复病毒进行筛选。结果显示,本研究成功建立了稳定的tk1-Vero细胞株;与143细胞相比,tk1-Vero细胞更适于病毒增殖及tk基因修复病毒的筛选;最后在tk1-Vero细胞中,利用HAT选择性培养液成功筛选出tk基因修复病毒。本研究表明,采用tk1-Vero细胞株建立的HAT选择性培养液筛选方法可以对tk基因修复病毒进行高效快速筛选。展开更多
基金The Nation "863" Program of China(2006AA02A226)The Joint Funds of National Science Foundation of China (U0632010)+2 种基金The State KeyLaboratory of Phytochemistry and Plant Resources in West ChinaChinese Academy of Sciences (O807B11211, O807E21211)"211 grant of MOE"
文摘RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.
基金This work was supported by the National High Technology Development Program.
文摘Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.
