摘要
目的构建表达人TGFβ1II型受体细胞外结合区和人IgG1Fc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RTPCR方法扩增目的基因TβRIIIgG1Fc,扩增产物纯化后克隆至测序载体pGEMTEasy,挑取阳性克隆酶切鉴定后测序;利用重组DNA技术,将TβRIIIgG1Fc基因亚克隆至逆转录病毒载体pSLXCMV中,重组质粒pSLXCMV/TβRIIIgG1Fc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、位置均正确无误,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pSLXCMV/TβRIIIgG1Fc,可望为肝纤维化的基因治疗提供有效途径。
Objective To construct recombinant human retroviral vector, which carries soluble TGFβ_1 Type II receptor (TRβII) gene. Methods The TβRⅡ-IgG1 F c gene was amplified by RT-PCR and the purified PCR products were ligated into t he sequencing vector pGEM-T-Easy. The white clones were selected and the recom bi nant plasmid was purified, which was further identified by restriction endonucle ases and was sequenced. TβRⅡ-IgG1 Fc gene was subcloned into retroviral vect o r pSLX-CMV by recombinant DNA technology. pSLX-CMV TβRⅡ-IgG1 Fc was packag d with PA317 and selected with G418 to obtain the positive clones, which was able to produce stable retrovirus. The integration and expression of TβRⅡ-IgG1 Fc ge ne in PA317 cells were identified by RT-PCR. Results Retroviral vector pSLX-CMV TβRⅡ-IgG1 Fc was contr ucted. PCR demonstrated that TβRⅡ-IgG1 Fc gene was integrated into the PA317 genome and expressed at the mRNA level. Sequencing, restriction digestion and PCR confirmed that the sequence, leng th and position of inserted gene were all correct. Conclusion pSLX-CMV TβRⅡ-IgG1 Fc was constructed successfully. It suggests that pSL X-CMV TβRⅡ-IgG1 Fc maybe is an effective gene therapy and will lay some fou ndation for clinical treatment of liver fibrosis.
出处
《胃肠病学和肝病学杂志》
CAS
2005年第3期230-234,共5页
Chinese Journal of Gastroenterology and Hepatology
