依地福新诱导重组Fas基因酵母的凋亡

Apoptosis Induced by Edelfosine in S. pombe Which Cloned Fas Gene

目的将人Fas基因转染至野生型粟酒裂殖酵母细胞中,并观察依地福新对重组酵母的促凋亡作用。方法抽提Jurkat细胞的RNA;RT-PCR制备Jurkat细胞的cDNA;PCR扩增人Fas基因;将人Fas基因克隆到pREP3X-HA质粒中构建pREP3X-HA-Fas穿梭载体,电转化穿梭载体到野生型粟酒裂殖酵母细胞中;应用Western blotting鉴定蛋白表达产物;应用依地福新诱导重组酵母产生凋亡应答。结果在33个酵母转化单菌落中,筛选到一个有表达活性的pREP3X-HA-Fas重组子,且依地福新能诱导此重组子的凋亡。结论成功构建pREP3X-HA-Fas穿梭载体并转入到相应的野生型粟酒裂殖酵母细胞中,且转化子具有表达活性。依地福新可能通过Fas诱导酵母细胞凋亡。

OBJECTIVE To obtain the human Fas gene and express in wild-type S. pombe,and observe the apoptosis induced by edelfosine in recombination yeast. METHODS Total RNA of Jurkat cells was extracted by TRIZOL reagent. First-strand cDNA of Jurkat cells was synthesized by RT-PCR. The Fas genes were amplified by PCR and cloned into plasmid and formed a relevant shuttle carrier. The pREP3X-HA-Fas shuttle carriers were transformed into wind-type S. pombe by Electroporation. The active recombination was identified by Western blotting and the apoptosis induced by edelfosine was observed. RESULTS An active recombination of pREP3X-HA-Fas was identified from 33 single clones of transformed S. pombe. Edelfosine induced apoptosis in transformed S. pombe. CONCLUSION The pREP3X-HA-Fas shuttle carriers have successfully been transformed into wild-type S. pombe; and the recombination of pREP3X-HA-Fas possess an expressed activity. Edelfosine could induce apoptosis by Fas in S. pombe.