携带Flag标签的人自噬相关蛋白4B的真核表达及功能鉴定

Eukaryotic Expression and Functional Characterization of Human ATG4B with Flag Tag

目的:构建携带Flag标签的人自噬相关基因4B(ATG4B)的真核表达质粒,获得Flag-ATG4B融合蛋白,并检测其与LC3的相互作用。方法:以本实验室保存的乳腺文库为模板,采用PCR技术扩增获得ATG4B序列,并将其插入pc DNA3.0-Flag载体构建成重组质粒,转染HEK293T细胞后用Western印迹检测融合蛋白的表达,用免疫共沉淀检测ATG4B与LC3的相互作用。结果:菌液PCR和重组质粒的双酶切结果及测序均表明重组质粒构建成功,Western印迹表明融合蛋白在HEK293T细胞中获得表达,免疫共沉淀结果表明表达的ATG4B能与LC3结合。结论:构建了pc DNA3.0-Flag-ATG4B真核表达质粒,表达的ATG4B蛋白对LC3的切割至关重要。

Objective： To construct recombinant eukaryotic expression plasmid of pcDNA3.0-Flag-ATG4B and de- tect the interaction between ATG4B and LC3. Methods： The fragment of human autophagy-related gene 4B （ATG4B） was obtained from a human mammary gland eDNA library by PCR and cloned into pcDNA3.0-Flag vec- tor. For expression analysis, HEK293T cells were transiently transfected with pcDNA3.0-Flag-ATG4B and analyzed by Western blot. For the interaction between ATG4B and LC3 analysis, HEK293T were transiently transfected with pcDNA3.0-Flag plus GFP-LC3, or peDNA3.0-Flag-ATG4B plus GFP-LC3, and analyzed by co-IP. Results： The pcDNA3.0-Flag-ATG4B was verified by PCR and double-enzyme cleavage. Western blot showed that the recombi- nant protein was successfully expressed. Co-IP showed that the recombinant protein can interact with LC3. Conclu- sion： The recombinant eukaryotic expression plasmid pcDNA3.0-Flag-ATG4B was successfully constructed. The pro- tein plays a critical role for cleave LC3, which lays a foundation for the future study of autophagy.