人Fas配体蛋白在大肠杆菌中的融合表达与应用

目的：在大肠杆菌中融合表达人Fas配体蛋白。方法：应用RT-PCR技术，从激活的人外周血淋巴细胞中提取总RNA，扩增Fas配体cDNA，克隆入PCR2.1载体，测序验证后，克隆入带有组氨酸盒的表达载体pQE-31，在大肠杆菌中表达，经亲和层析柱纯化后，用SDS-PAGE和Western blot鉴定表达产物。结果：表达的融合蛋白为人Fas配体，其相对分子质量（Mr)为40000。；经透析复性后，具有诱导Jurkat细胞凋亡的作用，用该蛋白分子免疫BALB/c小鼠制备抗血清，以间接ELISA检测了部分自身免疫病与肿瘤患者血清中可溶性的F 苛的含量。结果与进口试剂盒的灵敏性相似。结论：获得FasL单克隆抗体，深入研究FasL的应用提供了材料。

The expression and application of human Fas ligand in E.coli

AIM: To express recombinant human FasL molecule in E.coli. METHODS; RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2. 1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E. coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice. RESULTS: The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M, 40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits. CONCLUSION: The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.