中文摘要：

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LCgB序列，与PLA2G]O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。

英文摘要：

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.