摘要
目的:克隆自噬相关基因Atg5,在大肠杆菌中重组表达后制备抗Atg5多克隆抗体。方法:用RT-PCR方法从RAW264.7细胞基因组中克隆Atg5基因,连接至pQE80L原核表达载体后转化大肠杆菌DH5α进行诱导表达,SDS-PAGE及Western blot鉴定表达蛋白;目的蛋白纯化后,以100μg/kg纯化的蛋白免疫新西兰兔,制备抗Atg5多克隆抗体;提取RAW264.7细胞总蛋白,以制备的抗Atg5多抗进行Western blot反应,检测多抗的生物学活性。结果:克隆了Atg5基因,在大肠杆菌中表达了重组Atg5,SDS-PAGE分析显示表达产物相对分子质量与预期值一致,Western blot结果证明该产物具有较高的生物学活性,纯化蛋白免疫动物后制备了抗Atg5多克隆抗体。结论:在大肠杆菌中表达了重组Atg5,制备了抗Atg5多克隆抗体,为自噬的检测和研究提供了工具。
Objective： Autophagy-related gene Atg5 was colned and expressed in Escherichia coli DH5α for producing polyclonal antibody. Methods： After amplifing by RT-PCR from the genome of RAW264.7 cells, the Atg5 gene was subcloned into prokaryotic expression vector pQE80L and transformed to E.coli DH5α for expression. The specific expression product was identified by SDS-PAGE and Western blot. Rabbit was immunized with the purified protein for developing the polyclonal antibody against Atg5, then its bioactivity was detected by Western blot using the anti-Atg5 polyclonal antibody. Results： The Atg5 gene was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed that the recombinant protein was expressed as the predicated molecular mass and Western blot demonstrated that the expression product was of high bioactivities. The anti-Atg5 polyclonal antibody was obtained after protein purification and animal immunization. Conclusion： The recombinant Atg5 was successfully expressed, purified and the anti-Atg5 polyclonal antibody was developed, which provides an experimental basis for the detection and study of autophagy.
出处
《生物技术通讯》
CAS
2009年第4期482-484,共3页
Letters in Biotechnology
基金
国家自然科学基金(30872359)
陕西省自然科学基金(2007C263)
关键词
Atg5
原核表达
自噬
多克隆抗体
Atg5
prokaryotic expression
autophagy
polyclonal antibody
