摘要
目的:构建带Flag标签的自噬相关基因5(ATG5)的真核表达载体,获得Flag-ATG5融合蛋白。方法:以人乳腺文库为模板,采用PCR技术扩增人ATG5编码序列,将其插入pc DNA3-Flag载体,转染人胚肾293T细胞后,Western印迹检测其表达情况;将该重组质粒与ATG12表达质粒共转染293T细胞,通过免疫共沉淀法检测2个蛋白的相互作用。结果:Flag-ATG5真核表达质粒构建成功,转染293T细胞后融合蛋白获得表达,其相对分子质量约33×103;Flag-ATG5可与Myc-ATG12结合。结论:构建了带Flag标签的人ATG5真核表达载体,为进一步研究ATG5在自噬中的功能奠定了基础。
Objective: To construct and express the eukaryotic expression vector of human autophagy related gene 5(ATGS). Methods: Human ATG5 gene was obtained from human mammary gland cDNA library by PCR and cloned into vector pcDNA3-Flag. Human 293T cells were transfected with the recombinant plasmid Flag- ATG5 and the expression was detected by Western blotting. In addition, assay was applied to determine the inter- action between Flag-ATG5 and Myc-ATG12. Results: ATG5 eukaryotic expression vector was confirmed by dou- ble restriction enzyme digestion and inserted fragment sequencing. The expression of human ATG5 protein with rel- ative molecular of 33×10^3 in human 293T cells was identified by Western blotting. In addition, immunoprecipita- tion results showed that human ATG5 protein could interact with Myc-ATG12 in vivo. Conclusion: The eukaryotic expression vector of Flag-ATG5 was constructed and expressed in human 293T cells, which had laid foundation for the further study of the role of ATG5 in autophagy.
出处
《生物技术通讯》
CAS
2015年第5期645-647,共3页
Letters in Biotechnology
基金
国家自然科学基金(81472589
31100604)
北京市科技新星计划(Z141102001814055)
北京市自然科学基金(7132155)
军事医学科学院创新基金转化医学项目(ZHYX003)
关键词
人自噬相关基因5
克隆
真核表达
自噬
human autophagy related gene 5
cloning
eukaryotic expression
autophagy