铁蛋白基因Fth1的克隆及其在大鼠骨髓间充质干细胞中的表达

发布时间：2019-02-11 17:10

【摘要】：旨在构建编码大鼠铁蛋白重链多肽1(ferritin heavy chain 1,Fth1)的慢病毒,并检测其在大鼠骨髓间充质干细胞(RMSC)中的表达。PCR扩增大鼠Fth1基因,克隆至p Lenti-GFP-C慢病毒表达载体,包装慢病毒,感染RMSC细胞。荧光显微镜观察细胞内GFP荧光情况,Western blot检测Fth1的表达情况,WST-1试剂检测Fth1慢病毒感染组(RMSC-Fth1)和空载体组(RMSC-GFP)的细胞增殖活性。DNA测序结果表明,p Lenti-GFP-C-Fth1慢病毒载体构建成功。包装慢病毒感染RMSC细胞后,荧光显微镜下可见明显绿色荧光,表明感染成功。Western blot结果显示,实验组细胞裂解液中可检测到特异的Fth1表达条带。细胞增殖实验显示,与空载体组相比,过表达Fth1并不影响RMSC细胞生长。成功构建并包装携带大鼠Fth1基因的慢病毒,其能够成功感染RMSC细胞并表达Fth1蛋白,且对细胞增殖无明显影响。
[Abstract]:The aim of this study was to construct a lentivirus encoding rat ferritin heavy chain polypeptide 1 (ferritin heavy chain 1 and detect its expression in rat bone marrow mesenchymal stem cells (RMSC). Rat Fth1 gene was amplified by PCR. Cloning into p Lenti-GFP-C lentivirus expression vector, packaging lentivirus, infected RMSC cells. GFP fluorescence was observed by fluorescence microscope, Fth1 expression was detected by, Western blot, proliferation activity of Fth1 lentivirus infected group (RMSC-Fth1) and empty vector group (RMSC-GFP) was detected by WST-1 reagent. P Lenti-GFP-C-Fth1 lentivirus vector was successfully constructed. After packaging lentivirus was infected with RMSC cells, green fluorescence could be seen under fluorescence microscope. The results showed that the specific Fth1 expression bands could be detected in the cell lysate of the experimental group. Cell proliferation assay showed that overexpression of Fth1 did not affect the growth of RMSC cells compared with empty vector group. The lentivirus carrying rat Fth1 gene was successfully constructed and packaged. It can infect RMSC cells and express Fth1 protein successfully, and has no obvious effect on cell proliferation.
【作者单位】：浙江省农业科学院植物保护与微生物研究所;浙江大学医学院附属第二医院放射科;
【基金】：浙江省自然科学基金项目(LY15H180004) 浙江省公益项目(2015C32020)
【分类号】：Q78

【相似文献】