Stat1基因真核表达质粒的构建及其体外转染和表达检测

Construction eukaryotic expression plasmid of stat1 gene fragments and its transfection efficiency in vitro

目的克隆信号转导与转录活化子1(signal transducer and activators of transcription,STAT)基因片段并构建其真核重组表达质粒。通过研究Stat1 mRNA序列各组成部分与Stat1基因翻译调控的关系,探讨了Stat1蛋白翻译调控机制。方法采用DNA重组技术,将用SMART RACE法筛选出的Stat1基因片段克隆到pT-Adv载体,然后将其亚克隆到pFLAG-CMV-2真核表达载体上,构建Stat1 cDNA重组质粒。分别采用磷酸钙转染法、DEAE-葡聚糖转染法和脂质体介导转染法将其转染Hela细胞,并对3种方法的转染效率及各重组Stat1质粒mRNA和蛋白质表达的差异进行比较,以研究Stat1 mRNA结构对Stat1表达的影响。结果构建了CS,C3S,CS/3S,5SFC3S,5SFCS和CL/3L共6个重组Stat1质粒。通过比较发现,磷酸钙转染法、DEAE-葡聚糖转染法的转染效率明显低于脂质体介导转染法,转染的质粒均得到清晰的mRNA和蛋白质表达信号。用INF-α刺激转染5SFC3S重组Stat1质粒的Hela细胞,发现刺激组与空白组Stat1 mRNA转录水平和蛋白质表达量无明显差异,蛋白质合成效率无降低,此结果与野生型Stat1对INF-α刺激不同。结论将重组Stat1质粒转染Hela细胞后,在短暂表达条件下,重组Stat1质粒与野生型Stat1对INF-α刺激反应不同,其原因尚有待进一步研究。

[Objective] To clone Statl gene fragments, to construct eukaryotic recombinant expression plasmid and to determine whether Statl protein expression is displayed translational control and possible mechanisms of regulation. [Methods] Both 5- and 3- rapid amplification of Statl cDNA ends （RACE） were performed using SMARTTM RACE cDNA amplification technology. Then we used six CMV-Statl plasmid to transfect Hela cells by three different methods and compared the differences of expression of recombinant Statl plasmid and the transfection efficiency of each method. [Results] Recombinant plasmids （CS, C3S, CS/3S, 5SFC3S, 5SFCS, CU3L） were suc- cessfully constructed. We detected the expression of recombinant statl plasmids after transfection and got expression signal. The transfection efficiency of calciumphosphate mediated cotransfection and DEAE-Dextran transfection was lower than that of lipofectin transfection distinctively. There were not change between blank control and treated group with INF-α [ Conclusion] Recombinant plasmids have different stimulation reaction from INF-α, but the reason is not yet known.