APRIL siRNA表达载体构建、微流控芯片筛选及转染优化

Construction of APRIL isoform-specific siRNA expression vector, screening by microfluidic chip electrophoresis and transfection optimization

目的:构建增殖诱导配体(APRIL)异构体特异性siRNA质粒载体,利用微流控芯片电泳筛选APRIL异构体特异性siRNA表达质粒。方法:以高表达APRIL的人结肠癌细胞株SW480为肿瘤细胞模型,选择APRIL异构体特异性siRNA序列,微流控芯片电泳筛选siRNA表达质粒并经测序后转染SW480细胞,半定量RT-PCR法检测瞬时转染前后SW480细胞中APRILmRNA水平。结果:成功构建hOligo637、hOligo1450、hOligo1534、hOligo1750。微流控芯片电泳成功分辨出被双酶切的插入片段。瞬时转染SW480细胞后均产生特异性的mRNA抑制效应。结论:采用微流控芯片电泳成功筛选出APRIL为靶点的siRNA质粒,并可广泛应用于筛选siRNA质粒。

Objective：To construct APRIL isoform-specific siRNA expression plasmid and screen by microfluidic chip electrophoresis. Methods：Human colon carcinoma line SW480 with high-expressed APRIL gene as tumor cell model, APRIL isoformspecific siRNA sequences were selected and siRNA plamid vectors were screened by microfluidic chip electrophoresis. Then SW480 were transfected with the reformed plamids and APRIL mRNA expression level was detected by semiquantitative reverse transcription PCR （RT-PCR）. Results：HOligo637,hOligo1450, hOligo1534 and hOligo1750 were constructed successfully. The inserted oligos digested by two kinds of restriction enzymes could be distinguished in microfluidic chip. The special suppressing effects were produced after transient transfection with the reformed plamids. Conclusion： APRIL isoform-specific siRNA expression plasmids were screened by microfluidic chip electrophoresis successfully. It will be helpful to the study of APRIL in tumor cells.