应用LC3B-PLA2研究Atg4B对LC3B的切割作用  

LC3B-PLA2 for Measuring Cleavage Activity of Atg4B

作　　者：洪甜[1] 闫志风[2] 张立 徐小洁[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]中国人民解放军总医院妇产科,北京100853 [3]总参警卫局卫生保健处,北京100017

出　　处：《生物技术通讯》2017年第4期490-494,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81672602;81472589;81502264);北京市科技新星计划(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:构建带Myc标签的LC3B-PLA2基因的真核表达质粒,获得LC3B-PLA2融合蛋白,并应用LC3B-PLA2研究Atg4B对LC3B的切割作用。方法:以本实验室保存的乳腺文库为模板,PCR扩增获得LC3B序列,与PLA2G10拼接后插入pCMV-Myc载体,用构建的重组质粒转染HEK293T细胞,Western印迹检测融合蛋白的表达,用LC3B-PLA2与Atg4B共转染的方法检测Atg4B对LC3B的切割作用。结果:菌液PCR、重组质粒双酶切及测序均表明重组质粒构建成功,Western印迹结果表明融合蛋白在HEK293T细胞中获得表达,LC3B-PLA2真核表达蛋白能够应用于Atg4B对LC3B切割作用的研究。结论:构建了pCMV-Myc-LC3B-PLA2真核表达质粒,LC3B-PLA2融合蛋白在Atg4B对LC3B切割作用的研究中至关重要,为进一步研究Atg4B在自噬过程中的作用奠定了基础。Objective： To construct recombinant eukaryotic expression plasmid of LC3B-PLA2 and measure activity of Atg4B. Methods： The fragment of human LC3B gene was obtained from a human breast cDNA library by PCR and cloned into pCMV-Myc vector together with PLA2GIO. For expression analysis, HEK293T cells were transiently transfected with pCMV-Myc-LC3B-PLA2 and analyzed by Western blot. For the activity of Atg4B analysis, HEK293T cells were transiently transfected with peDNA3.0-Flag and pCMV-Myc-LC3B-PLA2, or pcDNA3.0-Flag- Atg4B and pCMV-Myc-LC3B-PLA2, and analyzed by Western blot. Results： pCMV-Myc-LC3B-PLA2 was veri- fied by PCR and double-enzyme cleavage. Western blot showed that the recombinant plasmid was successfully ex- pressed, and the recombinant protein can measure the activity of Atg4B. Conclusion： The recombinant eukaryotic expression plasmid pCMV-Myc-LC3B-PLA2 has been constructed, and it express protein which plays a critical role for measuring the activity of Atg4B, laying a foundation for the future study of the role of Atg4B in autophagy.

关 键 词：LC3B-PLA2 Atg4B 自噬 重组质粒 

分 类 号：Q78[生物学—分子生物学]