可溶性TGF-β_1Ⅱ型受体逆转录病毒表达载体的构建  

Construction of expression vector containing TGF-βRⅡ-IgG1 Fc genes

作　　者：高巍[1] 周永兴[1] 张惠中[2] 聂青和[1] 

机构地区：[1]中国人民解放军第四军医大学唐都医院全军感染病诊治中心,陕西省西安市710038 [2]中国人民解放军第四军医大学唐都医院中心实验室,陕西省西安市710038

出　　处：《世界华人消化杂志》2005年第6期760-765,共6页World Chinese Journal of Digestology

摘　　要：目的:构建表达人TGF—β1Ⅱ型受体细胞外结合区和人IgG1 Fc的融合蛋白逆转录病毒载体,为进一步肝纤维化的基因治疗奠定实验基础. 方法:以RT—PCR方法扩增目的基因TβRⅡ-IgG1 Fc,扩增产物纯化后克隆至测序载体pGEM—T—Easy,挑取阳性克隆酶切鉴定后测序;利用重组DNA 技术,将TβRⅡ-IgG1 Fc基因亚克隆至逆转录病毒载体pLXSN中,重组质粒pL(TβRⅡ-IgG1 Fc)SN在脂质体介导下转染PA317包装细胞,G418筛选,直至出现抗性克隆,扩大培养,测定病毒滴度. 结果:经测序、限制性酶切分析及PCR方法鉴定,载体插入基因序列、大小、位置均正确,并用PA317细胞进行包装、病毒滴度测定、筛选,建立具有较高滴度的感染性重组病毒产生细胞系. 结论:成功构建了重组质粒pL(TβRⅡ-IgG1 Fc)SN, 可望为肝纤维化的基因治疗提供有效途径.AIM: To construct recombinant human retroviral vector carrying soluble TGFβ1 Type Ⅱ receptor (TβR Ⅱ) gene. METHODS: The cDNA fragments of human TβRⅡ and IgG1 Fc were amplified by RT-PCR from total RNAs of peripheral blood mononuclear cells. The purified cDNA fragments were ligated and subcloned into vector pGEM-T-Easy to get the fusion gene of TβRⅡ-IgG1 Fc. The fusion gene was then subcloned into retroviral vector pLXSN. The obtained recombinant retroviral vector pL (TβRⅡ-IgG1-Fc) SN was transfected into PA317 cells and selected with G418. The stable expression of the TβRⅡ-IgG1-Fc fusion gene in positive clones was identified by RT-PCR. RESULTS: The restriction endonuclease digestion results and DNA sequencing indicated that the retroviral vector pL (TβRⅡ-IgG1 Fc) SN was successfully constructed. The TβRⅡ-IgG1 Fc fusion gene was integrated into the PA317 genome and expressed stably in the host cells. CONCLUSION: The target retroviral vector pL(TβRⅡ-IgG1 Fc) SN was constructed successfully. It provides an ef- fective tool for gene therapy and will lay a foundation for clinical treatment of liver fibrosis.

关 键 词：TGF-β1Ⅱ型受体 逆转录病毒表达载体 逆转录病毒载体 可溶性 重组DNA技术 PA317细胞 PCR方法 病毒滴度测定 基因治疗 肝纤维化 TβRⅡ 重组质粒 人IgG1 PLXSN 脂质体介导 限制性酶切 构建表达 融合蛋白 目的基因 产物纯化 

分 类 号：R346[医药卫生—基础医学]