可调控的胰岛素基因表达载体的构建及其体外表达研究

目的构建四环素调控的胰岛素基因表达载体 ,并研究其体外转移后的表达情况。方法通过基因重组技术构建胰岛素基因的四环素调控表达系统 ,用脂质体转移法导入成肌细胞 ,以不同浓度的强力霉素诱导 ,检测诱导后胰岛素原mRNA表达及胰岛素 /胰岛素原水平。结果RT PCR显示此基因能在真核细胞内表达。细胞培养液中胰岛素水平在加药 2 4h后开始增加 ,至第 7天仍在增加 ,撤药后迅速下降 ,至撤药后第 5天下降到基础水平。不同诱导药物浓度诱导的胰岛素产量明显不同 (P <0 .0 5 ) ,而细胞内和培养基中的胰岛素浓度无明显差异 (P >0 .0 5 )。结论单一四环素调控的胰岛素原表达系统能在真核细胞内表达 ,并呈现强力霉素浓度依赖性

Construction of a human insulin expression vector under the control of doxycycline and its expression in vitro

PurposeTo construct a single plasmid vector mediating doxycycline regulated human insulin gene expression. MethodsAn expression cassette of rtTAnls driven by human cytomegalovirus promoter(hCMV) and a recombined human insulin expression cassette driven by a reverse poly tetO DNA motif were cloned into a single plasmid vector (prTR tetO mINS). prTR tetO mINS and pLNCX were cotransfected into a myotube cell line (C2C12) and pLNCX vector was used as a control. After selection with G418, the transfected cells were induced with doxycycline at the concentrations of 0 μg/ml, 2 μg/ml and 10 μg/ml. Cells after different days of doxycycline incubation were harvested. RT PCR was used to determine expression levels of recombinant insulin mRNA at the three doxycycline concentrations on day 5. Insulin production in cell cultures and cell extracts was analyzed with human insulin radioimmunoassay(RIA) kits. ResultsImmune reactive insulin (IRI) was found to be increased at 24 hours of doxycycline incubation, and still increased at day 5. After being withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin tole mRNA levels were positively related to different levels of doxycycline. A 25 fold of increase in IRI was found to be against background expression.ConclusionHuman insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.