一种小鼠可溶性Fas cDNA的克隆与表达

为获得调节鼠Fas Fas配体系统诱导凋亡的作用 ,根据GenBank中小鼠Fas基因碱基序列 ,设计扩增可溶性Fas(solubleFas ,sFas)引物 ,用RT PCR方法从幼鼠胸腺组织中克隆出与人可溶性Fas类似的新的鼠sFas (FasC)cDNA序列 .该序列缺乏Fas基因的跨膜区片段 ,但不改变其阅读框 .采用定向克隆的方法将之插入pUC19中间载体 ,DNA序列测定证实该片段序列与预期序列完全一致 .利用亚克隆的方法将鼠FasC的cDNA片段克隆到真核表达载体pCA13上 ,构建出重组载体pCA13 FasC ,通过脂质体LF2 0 0 0转染至 2 93细胞 .RT PCR和Western印迹证实 ,鼠FasC在 2 93细胞获得高效表达 .凋亡诱导实验表明 ,鼠FasC的表达可阻断Fas诱导凋亡的作用 ,证实了所转染鼠FasC的生物活性 .

Cloning and Expression of the cDNA of a Murine Soluble Fas

In order to study the regulation of apoptosis induced by Fas Fas ligand (FasL) system, a soluble isoform of mouse Fas was cloned from immature mouse's thymocyte with the primers designed according to the Fas full cDNA sequence in the GenBank. This murine soluble Fas (Fas C) was similar to the human soluble Fas, which lacked the transmembrane region. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence. Then it was subcloned into the eukaryotic expression vector pCA13 to obtain the recombinant vector pCA13 FasC . By lipofectine(LF2000), pCA13 FasC was transfected into the 293 cells. RT PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine sFas could block the Fas induced apoptosis, which proved the existence of biological activity in the recombinant Fas C.