Hu Zhongbo, Zou Ping, Li Aixiang, Xiao Juan, Zhong Zhaodong, Liu Lingbo
Institute of Hematology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022.
J Huazhong Univ Sci Technolog Med Sci. 2002;22(3):186-9, 196. doi: 10.1007/BF02828175.
In order to regulate the apoptosis induced by Fas-FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full-length Fas cDNA sequence in the GeneBank. 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, which was used to construct the recombinant vector pCA13-FasC. By lipofectamine (LF2000)-mediated transfection, 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 Fas C could block the Fas-induced apoptosis, which confirmed the biological activity of the recombinant Fas C.
为调控Fas-FasL系统诱导的细胞凋亡，根据基因库中全长Fas cDNA序列设计引物，从未成熟小鼠胸腺细胞中克隆出小鼠Fas的可溶性异构体。将其定向插入中间载体pUC19。DNA测序证明其与预期序列一致。然后将其亚克隆到真核表达载体pCA13中，用于构建重组载体pCA13-FasC。通过脂质体（LF2000）介导的转染，将pCA13-FasC转染到293细胞中。RT-PCR和蛋白质免疫印迹表明，小鼠可溶性Fas C蛋白在293细胞中表达。细胞凋亡诱导试验表明，这种小鼠Fas C的表达可阻断Fas诱导的细胞凋亡，证实了重组Fas C的生物学活性。
