目的·研究SUMO化修饰对胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)蛋白的结构、稳定性及活性的影响。方法·建立体外表达纯化体系,获取可用于晶体筛选及活性检测的SUMO-1-TDG蛋白。通过晶体筛选、衍射数据收集及结构解...目的·研究SUMO化修饰对胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)蛋白的结构、稳定性及活性的影响。方法·建立体外表达纯化体系,获取可用于晶体筛选及活性检测的SUMO-1-TDG蛋白。通过晶体筛选、衍射数据收集及结构解析,分析SUMO-1-TDG的分子结构。利用蛋白热稳定性实验检测在SUMO化修饰前后TDG稳定性的变化。建立TDG活性测试体系,探讨SUMO化修饰对TDG活性产生的影响。结果·通过结构解析得到SUMO-1-TDG分子结构。稳定性、活性实验检测发现TDG蛋白熔解温度(Tm)值提高约16℃,催化活性提升约9.70%。结论·SUMO-1分子结合TDG对其进行修饰使得结合位点附近氨基酸形成分子间相互作用,并进一步参与调控TDG蛋白的稳定性及催化活性。展开更多
Objective To investigate the killing effect of herpes simplex virus thymidine kinase/gancyclovir(HSV tk/GCV)suicide gene therapy system on retinoblastoma(Rb)cells and the mechanism of bystander effect.Methods By using...Objective To investigate the killing effect of herpes simplex virus thymidine kinase/gancyclovir(HSV tk/GCV)suicide gene therapy system on retinoblastoma(Rb)cells and the mechanism of bystander effect.Methods By using liposome,pCMV/hytk IRES hrGFP plasmid was transferred into HXO Rb 44 cells.A fluorescence microscope was used to detect the transduction effeciency.The positive cell clones were selected by hygromycin and were named HXO Rb 44/tk.RT PCR was resorted to demonstrate the sucessful transduction and transcription of hytk gene in the HXO Rb 44/tk cells.The morphologic features and growth patterns of HXO Rb 44/tk were compared with those of HXO Rb 44.Then MTT assay was used to determined the killing effect of GCV on HXO Rb 44/tk and the mixture of HXO Rb 44/tk and HXO Rb 44 in different ratios('bystander effect').The mechanism of bystander effect was studied by the experiment of supernatant shifting.Results The transduction effeciency was 20%.530bp hytk gene strand was seen through HXO Rb 44/tk RT PCR.There were no differences in the morphologic features or the growth patterns between HXO Rb 44/tk and HXO Rb 44.HXO Rb 44/tk was more sensitive to GCV than was HXO Rb 44.The cytotoxicity of HXO Rb 44/tk was dose and time dependent.An obvious'bystander effect'was seen even with low proportions of HXO Rb 44/tk,but this effect disappeared when transferring GCV containing supernatant of HSV tk positive cells to the negative cells.Conclusions The transfer of the HSV tk gene into Rb cells followed by the administration of GCV could serve as a model for gene therapy for retinoblastoma,the'bystander effect'in HSV tk/GCV mediated gene therapy occurs by transfer of GCV metabolite from cell to cell through gap junction.展开更多
文摘目的·研究SUMO化修饰对胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)蛋白的结构、稳定性及活性的影响。方法·建立体外表达纯化体系,获取可用于晶体筛选及活性检测的SUMO-1-TDG蛋白。通过晶体筛选、衍射数据收集及结构解析,分析SUMO-1-TDG的分子结构。利用蛋白热稳定性实验检测在SUMO化修饰前后TDG稳定性的变化。建立TDG活性测试体系,探讨SUMO化修饰对TDG活性产生的影响。结果·通过结构解析得到SUMO-1-TDG分子结构。稳定性、活性实验检测发现TDG蛋白熔解温度(Tm)值提高约16℃,催化活性提升约9.70%。结论·SUMO-1分子结合TDG对其进行修饰使得结合位点附近氨基酸形成分子间相互作用,并进一步参与调控TDG蛋白的稳定性及催化活性。
文摘Objective To investigate the killing effect of herpes simplex virus thymidine kinase/gancyclovir(HSV tk/GCV)suicide gene therapy system on retinoblastoma(Rb)cells and the mechanism of bystander effect.Methods By using liposome,pCMV/hytk IRES hrGFP plasmid was transferred into HXO Rb 44 cells.A fluorescence microscope was used to detect the transduction effeciency.The positive cell clones were selected by hygromycin and were named HXO Rb 44/tk.RT PCR was resorted to demonstrate the sucessful transduction and transcription of hytk gene in the HXO Rb 44/tk cells.The morphologic features and growth patterns of HXO Rb 44/tk were compared with those of HXO Rb 44.Then MTT assay was used to determined the killing effect of GCV on HXO Rb 44/tk and the mixture of HXO Rb 44/tk and HXO Rb 44 in different ratios('bystander effect').The mechanism of bystander effect was studied by the experiment of supernatant shifting.Results The transduction effeciency was 20%.530bp hytk gene strand was seen through HXO Rb 44/tk RT PCR.There were no differences in the morphologic features or the growth patterns between HXO Rb 44/tk and HXO Rb 44.HXO Rb 44/tk was more sensitive to GCV than was HXO Rb 44.The cytotoxicity of HXO Rb 44/tk was dose and time dependent.An obvious'bystander effect'was seen even with low proportions of HXO Rb 44/tk,but this effect disappeared when transferring GCV containing supernatant of HSV tk positive cells to the negative cells.Conclusions The transfer of the HSV tk gene into Rb cells followed by the administration of GCV could serve as a model for gene therapy for retinoblastoma,the'bystander effect'in HSV tk/GCV mediated gene therapy occurs by transfer of GCV metabolite from cell to cell through gap junction.
