FAS抗原胞外区片段GST融合蛋白的产生

目的：获得重组ＦＡＳ抗原，用于抗体制备及进一步的功能分析。方法：用ＰＣＲ技术从完整的ＦＡＳｃＤＮＡ克隆上扩增其编码胞外区的部分，将ＰＣＲ产物直接克隆到ｐＧＥＭ－Ｔ载体系统，ＤＮＡ序列分析证实序列完全正确；用ＥｃｏＲⅠ和ＳａｌⅠ将ＦＡＳ胞外区片段切出，定向克隆到经同样酶切处理的ｐＧＥＸ－ＫＧ表达载体，转化大肠杆菌，经优化ＩＰＴＧ的诱导条件，获得了ＦＡＳ胞外区与谷胱甘肽－Ｓ－转移酶的融合蛋白高效表达；用亲合层析法从细菌粗提物中纯化了ＧＳＴ－ＦＡＳ融合蛋白，免疫家兔制备了抗ＦＡＳ抗体。结果：用重组ＦＡＳ为免疫原制备的抗体能诱导Ｕ９３７细胞产生细胞凋亡。结论：重组ＦＡＳ抗原和制备的抗体能用于对ＦＡＳ系统的功能研究

Production of recombinant FAS antigen and GST fusion protein

Objective: This research is aimed at expression of recombinant FAS antigen and preparation of anti FAS antibodies to lay the basis for functional study of FAS system. Methods: The DNA fragment encoding outer membrane part of FAS antigen was amplified by polymerase chain reaction(PCR) method. The PCR products were directly cloned into pGEM T system. DNA Sequencing analysis revealed a correct cDNA sequence of FAS antigen. The FAS DNA fragment was recovered and inserted into pGEX KG plasmid,then transformed into E.coli recipient cells. The recombinant clones were induced by appropriate IPTG concentration. A high level expression of recombinant FAS and glutathione S transferase fusion protein was obtained. Results: Recombinant FAS antigen was expressed and purified;anti FAS polyclonal antibodies were prepared;anti FAS antibodies could induce the apoptosis of FAS positive U937 cells. Conclusion: Recombinant FAS antigen and anti FAS antibodies can be used to analyze the functions of FAS system.