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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,克隆人PCR2.1载体。测序验证后,克隆人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40 000。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FasL的含量,结果与进口试剂盒的灵敏性相似。结论:获得人Fas配体蛋白,并制备出抗FasL抗血清,为进一步研制抗FasL单克隆抗体,深入研究FasL的应用提供了材料。
Objective: Fusion expression of human Fas ligand protein in Escherichia coli. METHODS: Total RNA was extracted from activated human peripheral blood lymphocytes by RT-PCR. The cDNA of Fas ligand was amplified and the human PCR2.1 vector was cloned. After sequencing, the recombinant plasmid pQE-31 with human histidine was cloned and expressed in Escherichia coli. After purification by affinity chromatography, the expressed product was identified by SDS-PAGE and Western blot. Results: The expressed fusion protein was human Fas ligand with the relative molecular mass (Mr) of 40 000. After dialysis refolding, Jurkat cells have the role of apoptosis. BALB / c mice were immunized with this protein to prepare antiserum. The indirect ELISA was used to detect the content of soluble FasL in sera of patients with autoimmune diseases and tumor. The result was similar to the sensitivity of imported kit. Conclusion: The human Fas ligand protein was obtained and the anti-FasL antiserum was prepared, which provided the material for further research on the application of FasL monoclonal antibody.